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ESSENTIAL
BIOCHEMISTRY
FOR MEDICAL STUDENTS
WITH PROBLEM-SOLVING EXERCISES

Edited by Alexander I. Glukhov,

Alexandra E. Gubareva

-GEOTAR-Media-
PUBLISHIHQ GROUP
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TEXTBOOK

ESSENTIAL
BIOCHEMISTRY
FOR MEDICAL STUDENTS
WITH PROBLEM-SOLVING EXERCISES

Edited by Alexander I. Glukhov,

Alexandra E. Gubareva

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Moscow
►GHOTAR-Mcdla-
PUbushing Group
2020
VUK 577.1(075.8) OI-VME-3473
EEK 28.707.2h73-1
E80

Editors:
4/exander JvanavicA G7ukhav — Doctor of Biological Sciences. Professor, Head of the
Department of the Biological Chemistry, I.M. Scchenov First Moscow State Medical
University (Scchenov University);
4/erandro E'vgcn/cvvja Gnfeareva — Candidate of Medical Sciences, Docent in the
Department of the Biological Chemistry, I.M. Scchenov First Moscow State Medical
University (Scchenov University).

E80 Essential Biochemistry for Medical Students with Problem-Solving Exercises:

textbook/cd. by A. 1. Glukhov, A. E. Gubareva. — Moscow : GEOTAR-Media,
2020. - 584 p.: ill. - DOI: 10.33029/9704-5650-7-Bl0-2020-1-584.
ISBN 978-5-9704-5650-7

This textbook is based on the Biochemistry lecture course of the Biological

Chemistry Department. I.M. Scchenov First Moscow State Medical University. The
textbook focuses on the modern basic principles of Biochemistry. This first edition
comprehensively covers the molecular basics of the human organism functioning in
normal conditions and during adaptation to the changing environment. The book also
provides modern insight inLo the molecular mechanisms of (he most common human
pathological conditions. Each topic includes the appropriate tests and situational
problems, allowing to study the material more interactively and train students in
clinical problem solving and diagnostic reasoning.
The book $Essential Biochemistry for Medical Students with Problem-Solving
Exercises* is intended for medical and pharmacy students studying in English,
Biochemistry postgraduate students, and teachers.

YAK 577.1(075.8)
BBK28.70Z2fl7.3-L

4// RjgAfs Reserved. A'o part q/rAis book, may fee reproduced, disfrifeiafed or copied in any/orm or
fey any means w/Aowf /Ac written permixs/on a//Ae original capvrigAf Ao/dcr. GE0T4R-.Media
Pirfe/isAfng Growp.

© FSAEI HE I.M. Scchenov First Moscow State

Medical University of the Ministry of Health of the
Russian Federation (Scchenov University), 2019
© GE OTAR-Media Publishing Group, 2020
ISBN 978-5-9704-5650-7 GEOTAR-Media Publishing Group, design, 2020
 CONTENTS

Au (hours .. ....... . .. ............ A .... A ........ 4

List of abbreviations 5
Chapter 1. The structure, properties and functions of proteins (EE Aora6A») 9
Chapter 2. Enzymes (E E KoroAAo) ...................................... 57
Chapter 3. Gene Expression and Protein Synthesis
(O.K Eawri/ora, A'/. /vaffov).................................................................................. 108

Chapter 4. Structure and functions of biological membranes.

Cell signaling pathways (AE CuAorewi, kA Go/c/acAe/do, .V.A Gttmdtova) .... 190
Chapter 5. Catabolism and cellular bioenergetics
(ZA ATiuchua, AK. Togirova).............................. __ ...___... 216
Chapter 6. Carbohydrate metabolism (S'. A EoroAjcvo) 253
Chapter 7. Lipid metabolism (AE GiiZwrcva, /I./.. Ka/irtkm)................................ 308

Chapter 8. Nitrogen metabolism (/../. Ewy) 394

Chapter 9. Molecular endocrinology (S. A tfarotanra) ............................... 483
Chapter 10. Tissue Metabolism. Liver Detoxification Function (/../. Ifcay) 554
 AUTHOURS

Alexandr 1. Glukhov — Doctor of Biological Sciences, Professor, Head of die

Department of the Biological Chemistry; I.M Sechcnov First Moscow State
Medical University (Sechcnov University)
Alexandra E. Gubareva — Candidate of Medical Sciences, Docent in the Department
ofthc Biological Chemistry, I.M. Sechcnov First Moscow State Medical University
(Sechcnov University)
Svetlana A. Vorobiova — Candidate of Biological Sciences, Docent in the Department
ofthc Biological Chemistry, I. M. Sechcnov First Moscow State Medical University
(Sechcnov University)
Elena V. Korobko - Candidate of Biological Sciences, Docent in the Department
ofthc Biological Chemistry; I.M. Sechcnov First Moscow State Medical University
(Sechcnov University)
Olga V. Samuilova — Candidate of Biological Sciences, Docent in the Department
of the Biological Chemistry, I.M. Sechcnov First Moscow State Medical
University (Sechcnov University)
Zaza A. Chuchua — PhD, The Heart Institute, Division of Molecular and
Cardiovascular Biology, Cincinnati Children's Hospital Medical Center. USA
Lyudmila 1. Usay — Candidate of Medical Sciences, Docent in the Department
of the Biological Chemistry, I.M. Sechcnov First Moscow State Medical
University (Sechcnov University)
Al Ila K. Tagirova — Candidate of Medical Sciences, Docent in the Department of
the Biological Chemistry, I M. Sechcnov First Moscow State Medical University
(Sechcnov University)
Alexandr L. Kalinkin — Candidate of Medical Sciences, Leading Researcher,
Department of Age Associated Diseases, University Hospital, M.V. Lomonosov
Moscow Stale University
Vera A. Goleochenko — Candidate of Biological Sciences. Docent in the Department
ofthc Biological Chemistry. I.M. Sechcnov First Moscow Stale Medical University
(Sechcnov University)
Konstantin L Ivanov — PhD. Docent in Molecular Biology University of Helsinki.
Finland
Natalia A. Gorenkova — Candidate of Biological Sciences, Docent in the Department
of the Biological Chemistry; I.M. Sechcnov First Moscow Slate Medical
University (Sechcnov University)
 LIST OF ABBREVIATIONS

aRC — ATP-binding cassette protein

AC — adcnvlyl cyclase
ACAI — acyl-CoA - cholesterol acyl transferase
Ach — acetylcholine
ACP — acyl carrier protein
ACTH — adrenocorticotropic hormone
ADH — antidiuretic hormone
ADLH — aldehyde dehydrogenase
AIDS — acquired immunodeficiency syndrome
ALA — y-aminolevulinatc
ALDH — acetaldehyde dehydrogenase
ALP — alkaline phosphatase
ALT — alanine aminotransferase
AP site — abasic site
apo A-l, apo Cl I, apo E, apo B100, apo IMS — apoproteins
AST — aspartate aminotransferase
ATF — activator transcription factor
ATPascs — adenosine 5’ triphosphatases
B9(Bc) — folic acid
BER — base excision repair
BFE — bi functional enzyme
BMI — body mass index
2,3 BPG — 2,3-Bisphosphoglyccratc
BVR — biliverdin reductase
Ca2+-ATPase — Ca2+-dependent adenosine 5’ triphosphatase
cAMP — cyclic adenosine monophosphate
CCP — common catabolic pathway
cDNA — complementary DNA
CETP — cholesterol ester transfer protein
cGMP — cyclic guanosine monophosphate
CK — creatine kinase
CM — chylomicrons
CoASH — coenzyme A
CoQ — coenzyme Q
COX — cyclooxygenase
CPS-I — carbamoyl phosphate synthetase 1
CPTI — carnitinc palmitoyl transferase 1
CRE — cAMP response clement
CREB — cAMP response element binding protein
CYP450 — cytochrome P450
Cyt — cytochrome
DAG — 1,2-diacy[glycerol
DHAP — dihydroxyacctonc phosphate
DHBP — dihydrobioptcrin
DHER — dihydro folate reductase
6 List of aDDreviaiiofls

I JIT — diiodoth.yroni.ncs
DNA pol — DNA polymerase
DNP — 2,4-di nitrophenol
DOPA — di hydroxyphenylalanine
DSB — DNA double-stranded break
DT — diphteria toxin
cEF — elongation factor
ETC — electron transport chain
FAD — flavin adenine di nucleotide
FH4 — tetra hydrofolatc
FMN — flavin mononucleotide
5-FU — 5-fluorouracil
fcjalNAc — N-acetylgalactosamine
GDH - L-glutamate dehydrogenase
GF — growth factor
GH — growth hormone
Gi — inhibitory GTP/GDP-binding protein
GLUT — glucose transporter
GOT - glutamate oxaloacclaLc transaminase
GPT — glutamate-pyruvate transaminase
Grt>2-protein — specific adaptor protein
Gs — activating GTP/G DP-binding protein
GSH — glutathione
GSSG — oxidized glutathione
H4BP(BH4) — tetrahydrob iopic rin
HAT — histone acetyl transferase
Hb — hemoglobin
HI JAC — histone deacetylase
HOL — high density lipoproteins
HIV - human immunodeficiency virus
HLGL — hepatic triacylglycerol lipase
HMG Co A — hydroxy-mcthylglutaryl Co A
H PETE - hydropcroxycicosatctracnioc acid
HSL - hormone sensitive lipase
HSV — herpes simplex virus
II) DM — insulin-dependent diabetes mcllitus
IDE — intermediate density lipoproteins
IMP — inosine monophosphate
IP3 — inositol 1,4,5-trisphosphate
IRS — insulin receptor substrate
a-KG — o-Kctogl uta rate
kB — kilobase
kIJA — kilodaltons
Km — Michaelis constant
LCA1 — lecithin cholesterol acyl transferase
LOH — lactate dehydrogenase
LDL — tow density lipoproteins
List of abbreviations 7

LPL — lipoprotein lipase

LT — leucoiricnc
MAtJ — monoacylglycerol
MAQ — monoamine oxidase
MCAD — medium chain fatty acyl CoA dehydrogenase
M E K-kinase — mitogen-activated protein kinase kinase
MEOS — microsomal ethanol oxidizing system
MGMT — methylguanine methyl transferase
miRNA — micro RNA
MIT — monoiodothyro nines
MMR — 1) NA mist match repair
mRNA — messenger RNA
MTasc — methyl transferase
MTP — microsomal triglyceride transfer protein
MTX — methotrexate
Na1, K -ATPase — Na', K*-dependant adenosine 5’ triphosphatase
NAD — nicotinamide adenine dinuclcotidc
NADP — nicotinamide adenine dinuclcotidc phosphate
NANA — N-acctyincuramintc acid)
NER — nucleotide excision, repair
NIDDM — non-insulin dependent diabetes me Ihtus
NMP — nucleoside monophosphate
NSAlDs — nonsteroidal anti — inflammatory drugs
NTP — nucleoside triphosphate
OA — oxaloacctate
ORE — open reading frame
PA — phosphatidic acid
PAHA — para-aminobenzoic acid
PAT — platelet-activating factor
PAH — phenylalanine hydroxylase
PAP — poly(A) polymerase
PCR — polymerase chain reaction
PDH — pyruvate dehydrogenase complex
PEP — phosphoenol pyruvate
PEPCK — phosphoenol pyruvate carboxykinase
PFK-J — phosphofructo kinase-1
PG — prostaglandin
Pl — isoelectric point
Pi — phosphate inorganic
P! — phosphatidylinositol
PIP, — phosphatidylinositol 4.5-bisphosphate
PKA — protein kinase A
PKG — protein kinase Ci
PKU — phenylketonuria
PLC — phospholipase C
PLP — pyridoxal phosphate
POMK — proopiomelanocortin
B List of aDDreviaiiofls

PPARs — peroxisome prolifcraior- activated receptors

PPi — pyrophosphate
PPP — pentose phosphate pathway
PRPP — I -phosphoribosyl-5-pyrophosphate
PTGS — post transcriptional gene silencing
PTM — post Lranslat io nal modification
qPCR — quantitative PCR
Ras — a G-protcin
rDNA — recombinant DNA
RF — releasing factors
RFLP — restriction fragment-length polymorphism
Ri — a primary messenger’s receptor, an inhibitor of adenylyl cyclase
system
RNA pol — RNA polymerase
RNAi — RNA interference
ROS — reactive oxygen species
rRNA — ribosomal RNA
Rs — a primary messenger’s receptor, an activator of adenylyl cyclase
system
RT-PCR — reverse transcription PCR
S — svedberg units
SAM — S-adenosyl methionine
sell) — severe combined immunodeficiency
siRNA — small interfering RNA
SNP — single nucleotide polymorphism
snRNAP — small nuclear ri bonne leop rotci n
SOD — superoxide dismutase
SOS — G D P-GT P exchanging factor
SR — scavenger receptor
SSB — DNA single-strand binding protein
TAG — triacyglycerols
TBP — TATA binding protein
TCA cycle — tricarboxylic acid! cycle
TF — transcription factors
Tgb — thyroglobulin
TPP — thiamine pyrophosphate
tRNA — transfer RNA
TX — thromboxane
Tvr-PK — tyrosine protein kinase
UCP-1 — uncoupling protein-L thermogenin.
UDP-GA — UDP-glucuronic acid
UGT — U DP-glucuronyl transferase
UTR — untranslated region
VLDL — very low density lipoproteins
Vrnax — maximal velocity
VZV — varicella-zoster virus
y-GT — Y-Gluiamyl transpeptidase
 Chapter 1
THE STRUCTURE, PROPERTIES
AND FUNCTIONS OF PROTEINS

11. Biological Functions of Proteins

12. Structures, Properties and Classification of Amino Acids
13. The Levels of Protein Structures: Primary, Secondary, Tertiary
14. Quaternary Structure of Proteins. Hemoglobin
15. Physical Chemical Properties of Proteins
16. Diseases Associated with Structure and Function Proteins Disorders

1.1 BIOLOGICAL FUNCTIONS OF PROTEINS

The structure and function of cel Is determine a large number of different molecules:
proteins, nucleic acids, carbohydrates. Proteins play a special role in the life of
the cell. First of a 11. the specific features of the structure and functioning of the cell arc
determined by a set of proteins synthesized in it. More than half of its dry substance
is attracted to the share of proteins inside the cell. In the human body there arc more
than 50,000 individual proteins. A set of proteins in Lhc body determines individual
specificity, the set of proteins in different cell types determines their morphological
and functional features.

Protein functions
► Structural hi net ion. Proteins arc directly involved in the construction of the
cell membranes and cytoskeleton (integral, sc mi-integral and surface proteins).
The substance of connective tissue and the extracellular matrix form proteins
collagen, elastin, keratin, proteoglycans.
► Enzymatic function All enzymes arc proteins. Enzymes catalyze
the transformations of various molecules in. the cells of the body. Enzymes
constitute more than 50% of all proteins.
► Receptor function. This function isthc selective bindingofhorniones, biologically
active substances and mediators on the surface of membranes or inside the cells.
► Transport function. Only proteins transport some substances in the blood. For
example, lipoproteins (lipid transfer), hemoglobin (oxygen transport), transferrin
(iron transport). Proteins transport cations of calcium, magnesium, sodium and
other ions into the blood.
10 Cnaptef 1. Tne structure, properties and functions of proteins

► Regulatory function. The regulation and coordination of metabolism in d ifFcrc n t

cells of the body arc carried out by hormones. Many hormones such as insulin
and glucagon are proteins, all pituitary hormones arc peptides or small proteins.
► Storage function. Animals and humans do not have specialized de pots of proteins,
but with prolonged fasting, muscle proteins, lymphoid organs, epithelial tissues
and the liver a re used.
► Contractile function. There arc several intracellular proteins designed to change
the shape of the celt and the movement of the cell ilselfor its organelles (tubulin,
actin, myosin)
► Protective function. Im mu nog tobul ins protect the body from the action ofbacleria.
toxins, foreign proteins, preventing the infectious process and maintaining the
stability of the body. The factors of the complement system also take place in the
body protection. Blood coagulation proteins work when the tissues arc damaged
(fibrinogen, prothrombin). Mechanical protection of mucous membranes and
skin provide collagen and proteoglycans.

IL STRUCTURES, PROPERTIES AND CLASSIFICATION OF AMINO

ACIDS
Proteins are high-molecular compounds and arc polymers formed from o-amino
acids linked together by peptide bonds. In nature, more than 300 different amino acids
arc known, but only 20 arc part of the proteins of humans and animals. Each amino
acid has a carboxyl group, an amino group in the alpha position (on the second carbon
atom) and a radical (side chain) that is specific for each amino acid (Fig. LI).

Side Chain

Fi^ 11. Basic amino acid formula

In aqueous solutions at neutral pH, amino acids exist as bipolar ions. All amino
acids (with the exception of glycine) contain an asymmetric carbon atom, therefore
they can exist as L- and II-stereoisomers (Fig. I.2). For the synthesis of human
proteins only L-a mi no acids arc used. In some protcinswithalong lifetime, L-isomers
can be converted to D-isomers.
All 20 amino acids in the human body differ in structure, size and physicochemical
properties of radicals (side chains). Amino acid radicals arc variable parts of a
polypeptide backbone and may contain various functional groups.
1.2. Structures, Properties and Classification of Amino Acids 11

L-alanine D-alanine

Fig. 12. Optical isomers of amino acids

► Polar (hydrophilic):
* Hydroxyl group-OH:
* Carboxyl group—COOH;
* Amino group -NH,_
* Imino group =NH2;
* Amide group -CO-NH,;
* Thiol group —SH.
► Non-polar (hydrophobic):
* Methyl group -CHa;
* Aromatic group.
To designate amino acids in proteins, their trivial names arc usually used. In
addition, for the convenience of recording the amino acid sequence of peptides and
proteins, their three-letter and one-letter designations arc used (Table LI).
Amino acids are classified according to the physicochemical properties of their
radicals. All amino acids can be divided into 4 groups
Amino acids can be divided into groups according to their ability to dissolve
in water. The solubility of amino acid radicals is determined by the polarity of the
functional groups. Polar groups attract water, non-polar repel it.
Amphotcricily is the main physicochemical property of amino acids. Amphoteric
means that the substance combines the properties of both acids and bases. In an
aqueous solution, amino acids simultaneously behave like acids — proton donors and
as bases — proton acceptors. Amino acids with polar negatively charged radicals have
an additional carboxyl group in the radical. Ai a physiological pH of 7.0, it dissociates
to form COO and H .The radicals of such proteins arc an ions. Ami no acids with polar
positively charged radicals have a second amino group in the radical. At physiological
pH, these groups arc positively charged. Radicals of such proteins arc cations.
 Chapter 1. Tnb structure, properties and functions of proteins

Table 1.1. C lassification of ami no acids on the c hemic al structure of their radicals

NONPOLAR +CHARGE
H „
’i’ 0 H,N‘-C-C*
H.M4-C-C*
RjN'-f-C*0 RsH4-i-C*° H.M'-C-C* ru °
h/
A
hi
ch^cHj SHj uC , 0 h
CH,
1
CH,
Glycine Alanine Valine Cy&Teine Praline I
NH
(flaw (VaLV| (CyS/C) [PrctfP)
C-NWj'
MH,' NH,
Lysine Arginine
t
HshC-OC*
L HjN'-C-C*
H3N+-C-C* h^w-c-c; CH, °
H,N+-C -C’
0 H^-CH ** MjN'-C-C*0
I ’
CH
*V
CH,
1
s
"S
dH, 0

6 1
CHjCHj

Leucine
SHj

Iso leucine
£H,

Hettlioriine Tryprophan Phenylalanine

tc
Histidine
[UhA) |M«Mi (W)
(H&H)

POLAR -CHARGE

H^JT-C-C*0
1 H.N*-C-C’
H.N4-C-C* HJN--C-C*0
HjN^-C-C* i H, 0
H^’-C-C*0 I J
^Hj,
1
CH,
1
CH,
I I
CH,

OH CH CHj
V
OH
NH^b A
HH,0 O
A
0
A
o o
Serine nweonine TyraEine Asparagine GjhjEamine Aspartic Acid Glutamic Acid
fSerffi) 0^ lAsfL-Ni (GWQ| (Asp®) (GUE)

Peptide bond. The structure and properties of peptides

Amino acids can covalently bind to each other using pcpii.de bonds. A peptide bond
is formed between the a-carboxyl group of one anti no acid and the a-amino group of
another, i.c. is an amide bond. Wien a peptide bond is formed, a water molecule is
split off (Fig. 1.3).
The amount of amino acids in the composition of the peptide can vary. Peptides
containing up to 1.0 amino acids arc called oligopeptides. Peptides containing more
than 10 amino acids arc called polypeptides. Polypeptides containing more than
50 amino acid residues arc called proteins.
The monomers of amino acids that make up the protein arc called amino acid
residues. Amino acid residue having a free amino group is called N-terminal and is
written on the left. Amino acid residue having a free carboxyl group is called C-terminal
and is writ ten on the right (Fig. 1.4). Amino acid residues in a polypeptide chain arc
numerated from the N-terminus. A chain of repealing amino acid residues without
radicals is called a peptide backbone.
1.2. Structures, Properties and Classification of Amino Acids 13

R R R

O II □ I
II I II

h3n'— c;—e- n — e—c n —c — COO

4
f
f
II
Hi H
\l
]HH

I ’eptide bonds

Pqptide
backbone

Fig. U. Peptide bond formation

CH-j—C —NH—CH—C- NH —CH —COs"

Tyf - Gly - Gly - Pn.fi - Met

N — terminal C — terminal
a mino acid amino acid

Fig. 1.4. Formula of penlapeptide Tyr-Gty-Gty-Pre-Mel

The peptide bond formed by the imino group of the proline di Ilers from other
peptide bonds, since the nitrogen atom of the peptide group is associated not with
hydrogen, but with a radical (Fig. I.5).

h2N — CH — CO — NH —CH—CO—N—CH— COCH

CH
I / \
H HPC CHj
h3c^ "'oh X /
ch2

Fig. 1.5. Formation of a peptide bond between Threonine and Proline

14 Chapter 1. Tnb structure, properties and functions of proteins

The pc pride bond is a strong covalent bond, It has a partial double bond character.
The peptide bond's length is [css than a single bond, it is a rigid (planar) struct Lire,
and rotation around it is difficult. But since, in addition to the peptide, there arc other
bonds in the protein, the chain of amino acids is able to rotate around the main axis,
which gives proteins a different conformation (the spatial arrangement of atoms). All
atoms in the peptide group arc in the same plane, while the atoms H and 0 arc located
on opposite sides of the peptide bond (Fig. 1.6 A). The oxygen and hydrogen atoms
in the peptide group have the ability to form hydrogen bonds with the oxygen and
hydrogen atoms of other peptide groups (Fig. 1.6 U). Amino acid radicals in relation
to the axis of the peptide C-N bonds arc on opposite sides, in the trans-position
(Fig. 1.6 C).

RO RO
B. I II ll

HO O
I I 1
— N — CH — C—N—CH — C-
I I I
R HR

Rg. 1.6. Properties of tne peptide bond

These properties of the peptide bond determine the ability of amino acids to
interact with each other within one protein, as welt as with, other proteins. Peptide
bonds arc very strong and under physiological conditions they do not spontaneously
break. In laboratory conditions, the peptide bonds arc hydrolyzed in the presence of
concentrated hydrochloric acid at 105° C within a day. In living organisms, peptide
bonds in proteins are destroyed with the help of special proteolytic enzymes —
proteases. To delect proteins and peptides in a solution, the color biuret reaction is
used.

The biological role of amino acids and peptides

Amino acids arc the building blocks of protein molecules, but their functions arc
not limited to this. Amino acids such as histidine, tryptophan. glutamic acid, tyrosine
arc the sources for the formation of neurotransmitters in the CNS (respectively.
12. Structures. Properties and Classification of Amino Acids 15

histamine, serotonin, gamma-aminobutyric acid, dopamine and noradrenaline >.

and glycine and glutamic acid arc neurotransmitters themselves. The amino acid
methionine is necessary for the synthesis of phosphatidylcholine, one of the main
components of cell membranes. .Amino acid tyrosine is completely included in the
composition of thyroid hormones (thyroxin. triiodothyronine) and adrenal medulla
(adrenaline, norepinephrine). Certain amino acids are necessary for the synthesis
of purine and pyrimidine which are the precursors of nucleic acids synthesis. Some
antino acids are used for the synthesis of low molecular weight biologically important
compounds (creatine. carnitine, carnosine, anserine, etc.).
A number of hereditary and acquired diseases, accompanied by serious
problems in the development of the organism, such as cystinosis, homocysicincmia.
leucinosis, tyrosincmia. etc., arc associated with metabolic disorders of anti no acids.
Phenylketonuria is the most famous example of metabolic disorders of amino acids.
Phenylketonuria, also called PKU. is an inherited disorder caused by a defect in the
gene that encodes the enzyme needed for phenylalanine metabolism. This eventually
leads to serious health problems.
One of the most common peptides with protective properties is tripepride
glutathione. Glutathione (GSH) is often referred to as the body's master antioxidant.
Reduced glutathione (GSH) is a linear tripeptide of L-glutaminc. L-cysteinc. and
glycine. The molecule has a sulfhydryl (SH) group on the cysteinyl portion, which
accounts for its strong electron-donating character. As electrons arc tost, the molecule
becomes oxidized, and two such molecules become linked (dimerized) by a disulfide
bridge to form glutathione disulfide or oxidized glutathione (GSSG). This linkage is
reversible upon re-reduction.

NH2—CH—CH2—CH2—C—NH—CH—C—NH—CH2—COOH

CHa CH2 —SH

Some ami no acids a nd severs I peptides arc important human hormones. Hormones,
in general, arc biological molecules used in multicellular organisms io direct and
regulate biological processes, such as growth, reproduction and metabolism. A peptide
hormones are chains of amino acids, which serve as a biological communication
molecules. Peptide hormones have a short half-life, meaning they break apart quickly.
This allows organisms to use peptide hormones to direct processes quietly and
efficiently, without the signal lingering fora long time.
The antino acid-derived hormones arc relatively small molecules derived from
the amino acids tyrosine and tryptophan. If a hormone is antino acid-derived, its
chemical name will end in «-inc*. Examples of amino acid-derived hormones include
epinephrine and norepinephrine, which arc synthesized in the medulla of the adrenal
glands, and thyroxine, which is produced by the thyroid gland. The pineal gland in the
brain makes and secretes melatonin, which regulates sleep cycles. The formulas antino
acid-derived hormones arc below:
16 Cnaptef 1. Tn e structure, properties and functions of proteins

Tryptophan Melatonin

The structure of peptide hormones is that of a polypeptide chain (chain of amino

acids). The peptide hormones include molecules that arc short polypeptide chains,
such as antidiurelic hormone and oxytocin produced in the brain and released into the
blood in the posterior pituitary gland. This class also includes small proteins, such as
growth hormones produced by the pituitary. Secreted short proteins, such as insulin,
are stored within, vesicles in the cells which synthesize them. They arc then released
in response to stimuli (c.g.r as high blood glucose levels in the case of insulin ). Amino
acid-derived and polypeptide hormones arc water-soluble and insoluble in lipids.
These hormones cannot pass through plasma membranes of cells; therefore, their
receptors are found on the surface of the target cells.
The group of peptides that affect vascular tone (vasoactive) includes bradykinin,
kallidin and angiotensin. The first peptide contains 9 amino acid residues, the
second — 10, and the third — 8. All of them arc synthesized from inactive protein
precursors as a result of the post-translational modification process. Peptides can
regulate the processes of digestion, for example, gastrin, cholecystokinin. Peptides
that regulate appetite arc. for example, leptin, b-endorphins.
Peptides, called enkephalins, or opiate peptides, arc found in the brain tissue and
perform an analgesic effect similar to that of opium substances. Another type of opiate
(anesthetic) peptides has also been found in the brain — and they are called endorphins.
These longer peptides (from 13 to 30 a..a.) got their names for the analgesic effect,
similar to the effect of morphine. They have a more complex physiological effect and
have not only an anesthetic effect, but also affect behavior.
Many toxins arc pepLidcs. For example, the toadstool (Amanita phalloidcs)
contains peptide toxins a man i tin and p hallodin. They are contained in these
mushrooms in high concentrations. The lethal dose lor humans is about 5-7 mg,
that is, one or two eaten by the fungus can cause death. All toxins of this type arc
cyclic peptides. Amatoxins cause a violation of RNA synthesis in celts, and phalloidin
violates the integrity of the membrane of the liver cells — hepatocytes. Peptide
toxin from bee venom (Apis mclifera) apamin, a linear peptide of 18 a.a, affects the
1.3. The Levels of Protein Structures: Primary, Secondary, Tertiary 17

functioning of calcium channels in membranes, mellitin — a peptide of 22 a.a. —

causes ionic conductivity in membranes, and the third — MSD-peptide causes allergic
and inflammatory reactions. Peptide toxins from snake venoms can be attributed to
protein substances by the number of amino acids, but they traditionally suggest the
presence of peptides. These toxins, as a rule, act on the membranes of nerve cells
or axons, disrupt their normal functioning. However, in low concentrations, toxins
and poisons arc used as effective drugs against a number of diseases associated with
neuromuscular disorders.

13. THE LEVELS OF PROTEIN STRUCTURES: PRIMARY, SECONDARY,

TERTIARY
Peptide chains contain hundreds and thousands of amino acid residues linked
by strong polypeptide bonds. Due to intramolecular interactions, proteins form
a specific spatial structure, called protein conformation. The linear amino acid
sequence in a protein , determines the construction of a three-dimensional spatial
structure. There are 4 levels of the spatial organization of proteins: primary,
secondary, tertiary and quaternary structures ( Fig. 1.7). There arc general rules by
which they arc formed.

Heme

Hydrogen |*. polypeptide

(c) Tertiary structure

n pctypeptide
ft polypeptide

(a) Primary structure

(U) Secondary structure (d) Quaternary structure

Fig. 1.7. Protein organization levels

18 Chapter 1. Tnb structure, properties and functions of proteins

Primary structure
/Imino acid residues in the peptide chain arc not randomly located, but arranged
in a specific order. The linear sequence of amino acid residues in a protein is called the
primary structure of (he protein (Fig. 1.8).

Fig. 1.8. Primary structure of We protein

The primary structure of proteins, i.c. the ami no acid sequence in it is programmed
by the nucleotide sequence in the DMA. The deletion, insertion, replacement of a
nucleotide in DNA leads to a change in the amino acid composition and. therefore,
the structure of the synthesized protein. If a change in the amino acid sequence is
not lethal, but adaptive oral least neutral, then the new protein can be. inherited and
remain in the population. Asa result, new proteins appear with similar functions. This
phenomenon is called, protein polymorphism.
For example, there are about JOO different types of hemoglobin, some of them arc
necessary al different stages of ontogenesis: for example. HbP — embryonic, formed
in the first month of development, HbF — fetal, necessary in the later stages of fetal
development, HbA and HbA2 — adult hemoglobin. The diversity is provided by the
polymorphism of globin chains: there are 2g and 2r chains in hemoglobin P. 2a and
2y chains in HbF. 2a and 20 chains in HbA. and 2a and 2d chains in HbA2. Proteins
ofthe main histocompatibility complex provide tissue transplantation incompatibility.
They have extremely high polymorphism, in general, there arc several million alleles
of those proteins. Due to this diversity, each person has an almost unique set of alleles.
In addition, many genetic diseases result from the amino acid sequence violation,
of proteins. Information about the primary structure of a normal and mutant protein
is needed to diagnose and predict the development of a disease.

Secondary structure
The secondary structure of a protein is a spatial structure resulting from in teracl Ions
between the functional groups that make up the peptide backbone. The secondary structure
1.3. The Levels of Protein Structures: Primary, Secondary, Tertiary 19

is formed only with the participation of hydrogen bonds between peptide groups: the
oxygen atom of one group reacts with, the hydrogen atom of the second, while the
oxygen of the second peptide group is bound to the third hydrogen, etc. (Pig. I.9).
O RO
II ] II
-N — CH — C — N—CH — C — N—
I I
HR H H

R O 0
|
II
-N — CH —C — N— CH — C — N—
| I I
H H R H
■
RO O
I II II
«N —CH—C —N —CH —C —N—
I
H
III
H R ft

Fig. 1.9. Hydrogen bonds between peptide backbone groups form a secondary protein structure

When the secondary structure is formed, the peptide accepts the conformation
with the largest number of bonds between the peptide groups. The type of secondary
structure depends on the stability oft he peptide bond, the mobility of the bond between
the central carbon, atom and the carbon of the peptide group, the size of the amino
acid radical. All of this, together with the amino acid sequence, will subsequently lead
to a strictly defined configuration of the protein. I n this case, peptide chains can form
regular structures of two types: a-helix and the P-shcct.
I none protein, asa rule, both structures a re simultaneously present, but indifferent
proportions. In globular proteins, the a-helix predominates, in fibrillar proteins, the
p-structure prevails.

aHeiix
The most common form of the secondary structure is the a-hclix (the polypeptide
chain seems to be twisted clockwise on an imaginary cylinder, which is due to the
L-amino acid composition of natural proteins). Al each turn (step) of the helix there
arc 3.6 amino acid residues, the helix pilch is 0.54 nni per turn, and one amino acid
residue isO.IS nm(Fig. 1.10).
Not all globular proteins arc helical across the entire length of the polypeptide
chain. In the protein molecule, the a-helical regions alternate with the linear ones.
Practically all atoms of the oxygen and hydrogen peptide groups arc involved in the
formation of hydrogen bonds. Since all the hydrophilic groups of the peptide core arc
occupied, the hydrophilicity a-helix (the ability lo form hydrogen bonds with water)
decreases, and the hydrophobicity increases.
20 Cnaptef 1. Tn e structure, properties and functions of proteins

Fig 1.10. The secondary structure of proteins in the term of a-hdix

The a-hclix is a ver)' stable conformation of the peptide backbone. The amino
acid radicals arc on the outside of the a-helix and arc directed away from the peptide
backbone. They do not participate in the formation of hydrogen bonds characteristic
of the a-helix, bin some may disrupt its formation, proline and hydroxyprolinc cause
chain bending, for example, in collagen.

fkSbeet
A p-shect is formed by the formation of hydrogen bonds between the atoms of
the peptide groups of the linear regions of one polypeptide chain. making bends or
between many different polypeptide chains (Fig. I JI). It looks like a folded sheet. In
this way of laying the protein the molecule makes a snake-shaped form, the remote
segments of the chain arc going close to each other. As a result, peptide groups
that previously were remote amino acids of the protein chain arc able to interact by
hydrogen bonds.
The orientation of the reactive sites may be parallel (i.c. the direction of N-terminal
to C-terminal ends is the same) or anti parallel where chains go in the opposite
direction (Fig. 1.12). Such sites of one protein interacting with each other can be from
two to five.
1.3. Trie Levels of Protein Structures: Primary, Secondary, Tertiary 21

Fig. 1.11. The secondary structure of proteins in the form of |Tsheet

The p-shests

Fig. 1.12. Parallel and antiparallel [-L sheets

22 Cnaptef 1. Tn e structure, properties and functions of proteins

Irregular secondary structures

Some parts of the protein arc ordered but do not form any regular structures. They
should not be confused with a random coil, an unfolded polypeptide chain lacking any
fixed three-dimension al structure. They are represented by loop-1 ike and ring-shaped
structures having a less regular packing than the a-helix and p-sheet described above.
However, they do not vary so much from one protein molecule to another. In each
individual protein, they have their fixed conformation, determined by the amino acid
composition of this chain segment and its surrounding regions.

Tertiary structure
The tertiary structure of proteins is a three-dimensional spatial structure formed
due to interactions between amino acid radicals, which can be located at a considerable
distance from each, other in the polypeptide chain. Secondary structures of proteins
often constitute distinct domains. A domain is the basic unit ofst rue lure and function.
Tertiary structure describes the relationship ofdilfercni domains to one another within
a protein. Pour types of chemical bonds arc involved in the formation of the tertiary
structure: hydrophobic, ionicT hydrogen and disulfide (Fig. 1.13).

Hydrophobic inreractiotis
{clustering of hydrophobic
groups away from water)
and van der Waals
interactions
Ch2 —------- - Polypeptide
zCH3 backbone
Hydrogen CH
bond

— CHj —S—S“CH2 —

Disulfide bridge

— Ch2—CHj—CH2— Ch2— Nl-V

Ionic bond

Fig. 1.11 Types at chemical bonds are involved in the formation of the tertiary structure

► Hydrophobic interactions: Hydrophobic interactions occur between non-polar

hydrophobic radicals of amino acids that formed the protein. The polypeptide
chain of a protein tends to take an energetically stable form, characterized by a
minimum office energy. Therefore, hydrophobic amino acid radicals tend to unite
1.3. Trie Levels of Protein Structures: Primary, Secondary, Tertiary 23

within the globular structure of water-soluble proteins. Between them, so-called

hydrophobic interactions arise, as well as van dor Waals forces between closely
adjacent atoms. Asa result, a hydrophobic core forms inside the protein globule.

Phe-Phe Hydrophobic interactions

► Ionic bonds: Ionic bonding can occur between negatively charged (anionic)
carboxyl groups of aspartic and glutamic acid radicals and positively charged
(cationic) groups of lysine, arginine and histidine radicals.

peptide
chain CH3
l z Glu-LyS
CHa -R-C^o- Hahr— R—
CHg nh2
1
CH2 LyS 1 Glu-Arg
ionic pond ■ rn ‘“-Hghr—C — NH—R —
: 1 :
I 1
i 1 n n Asp-Hs
o-
l HNt NH
V
4=O.J
CH2 Asp

► Hydrogen bonds: Hydrogen bonds occur between hydrophilic uncharged groups

—OH, —CONHp —SH, and any other hydrophilic groups.

— R—O—H-------- O=C—R— Ser-Asp

peptide Q|_| a.a. radicals

chain

— R—C=O*ii™h — O— R—;
s— R ■—■ N — H ■■■■■1111*“ O-— R —1 H — R— GyS-Glu
I I
H H
hydrogen Ponds Tyr-Lys
24 Cnaptef 1. Tn e structure, properties and functions of proteins

► Disulfide bonds: The covalent disulfide bond is formed between two -SH groups
of cysteine radicals located in different places of the polypeptide chain. Disulfide
bonds can. stabilize the spatial structure of a single polypeptide chain or link lwo
chains together, such as in an insulin molecule.

ww NH — CH — CO 'xyx/w
peptide 1
chain CHg

: s ■
disulfide
.oJi
bond
S ■

CHj
I
'wvv NH — CH — CO "ww

All proteins with the same primary structure and under the same conditions
acquire the same conformation, which determines their function. The functionally
active protein conformation is called the native structure. Hydrophobic, ionic and
hydrogen bonds arc weak bonds, therefore their breaking is possible even under
physiological conditions. This fact ensures the conformational lability of proteins,
i.c. they arc capable of small changes due to the breaking of some weak bonds and the
formation of others. Protein conformation can change with changing the chemical
and physical properties of the medium, as well as when interacting with other
molecules. Conformational changes play a huge role in the functioning of proteins
in a living cell
The breaking of a large number of weak bonds leads to the destruction of the native
conformation of the protein. The loss of the native conformation is accompanied by the
loss of the specific function of the protein. This process is called protein denaturation.
When denaturation occurs, an occasional break of weak bond's happen and protein
molecules acquire a random conformation.
Initially the weakest bonds are lorn and when conditions arc tightened, stronger
ones arc also broken. Therefore, al first, the quaternary, then the tertiary and secondary
structures arc lost. Denaturation docs not break the peptide bonds, i.c. the primary
structure of the protein is not disturbed.
Denaturation may be reversible., if restoration of the protein-characteristic structure
is possible (for example, membrane receptors): or irreversible, if the restoration of
the spatial configuration of the protein is impossible. Irrcvcsible denaturation usually
occurs when a large number of bonds arc broken, for example, when eggs arc boiled.
If the protein has undergone reversible denaturation, then when normal conditions
of the environment arc restored, it is able to completely restore its structure and,
accordingly, its properties and functions. The process of restoring the protein structure
after denaturation is called maturation (Fig. 1.14).
1.3. Trie Levels of Protein Structures: Primary, Secondary, Tertiary 25

Native state; Unfotdfed slate; inactive. Native, catalytically active

cate tyfcany active. DiaJfide cross -links reduced stele. Discitoe cross-links
to yield Cys residues. correctly re-formed.

Fig. 1.14. Denaturation and renaturatiom of riboituclease

Proteins can be denatured al high temperatures (over 50 *C), by vigorous shaking

of the protein solution, organic substances (alcohol phenol urea), acids and alkalis,
heavy metal salts, detergents. The most famous detergent is soap. In medicine,
denaturing agents are used to sterilize instruments, materials, and as antiseptics.
A protein domain is an element of the tertiary structure of a protein, which js a
fairly stable and independent substructure of a protein, the folding of w hich passes
independently of the other parts. The domain usually includes several elements of
the secondary structure. Domains similar in structure arc found not only in related
proteins (for example, hemoglobins of different animals}, but also in completely
different proteins. Domainscan perform different functions and undergo folding into
independent compact globular structural units interconnected by flexible sections
within a protein molecule (Fig. 1.15).

a «iA|l p

Fig. 115. Examples of protein domains classified by CATH (class, architecture, topology. homology)
26 Cnaptef 1. Tn e structure, properties and functions of proteins

Quite often, domains arc assigned separate names, since their presence directly
affects the biological functions performed by the protein, for example, the Ca2‘
binding domain of calmodulin, a homcodomain responsible for binding to ONA in
various transcription factors. Different domains in the protein can move relative to
each other when interacting with the ligand, which facilitates (he further functioning
of the protein.
The formation of the three-dimensional structure of the protein in the cell
is the most important process, since its biological function depends on the spatial
structure of the protein. The process of packing the polypeptide chain into the correct
spatial structure is called protein folding. The concentration of proteins in a cell is
very high, so an abnormal protein conformation may occur. For many proteins with
high molecular weight and complex spatial structure, folding occurs with the help of
special chaperone the participation proteins. Chaperones isolate the protein from the
environment and allow it to accept the native conformation.

Protein functioning
Each protein with a unique primary structure and conformation has a unique
function. Proteins perform many different functions in a cell. A prerequisite for the
functioning of a protein is the binding of a not her sub stance called a ligand. Ligands can.
be both low molecular weight substances, such as metal ions, small organic molecules
and macromolecular substances, such as other protein molecules. The interaction of
the protein with the ligand is highly specific, which is determined by the structure of
the protein site, called the active she (active center) of the protein.
The active site (active center) of proteins is a specific part of a protein molecule,
usually located in its recess pocket*), formed by the ami.no acid radicals collected
in a certain spatial region during the formation of the tertiary structure and capable of
complementary binding to the ligand (Fig. 1.16).

Fig. 1.16. The active site of me protein and its imeradion with me ligand

In the linear sequence of the polypeptide chain, the radicals forming the active
center can be located al a considerable distance from each other. The high specificity
of protein binding to the ligand is ensured by the complementary structure of the
active center of the protein to the ligand structure.
1.3. Trie Levels of Protein Structures: Primary, Secondary, Tertiary 37

Complementarity means the spatial and chemical correspondence of interacting

molecules. The ligand must have the ability to enter and spatially coincide with the
conformation of the active center. This coincidence may be incomplete, but due to
the conformational lability of the protein, the active center is capable of small changes
and «fits* into the ligand. In addition, between the functional groups of the ligand and
the radicals of the amino acids forming the active center, bonds must arise that hold
the ligand in the active center. The bonds between the ligand and the active center of
the protein can be both non-covalcnt (ionic, hydrogen, hydrophobic) and covalent
(Fie- 1-J7)

Fig. 117. The interaction of protein with a ligand in me active site. A and B are incomplete interaction.
C is complementary interaction. L-ligaiM

The unique properties of the active center depend not only on the chemical
properties of the amino acids forming it, but also on their exact mutual orientation
in space. Therefore, even minor violations of the general conformation of the protein
as a result of point changes in its primary structure or environmental conditions can
lead to a change in the chemical and functional properties of the radicals that form the
active center, disrupt the binding of the protein to the ligand and its function. During
denaturation, the active center of proteins is destroyed, and their biological activity
is lost. Often the active center is formed in such a way that the access of waler to the
functional groups of its radicals is limited, i.e. conditions arc created for the binding
of the ligand to amino acid radicals. In some eases, the ligand is attached to only one
28 Cnaptef 1. Tn e structure, properties and functions of proteins

of the atoms with a certain reactivity, for example, the addition ofO2 to the iron ion of
myoglobin, or hemoglobin.
The main property of proteins that underlies their functions is the selectivity of
specific ligands to attach to certain sections of the protein molecule.
There arc ligands that can change their chemical structure upon attachment to the
active center of the protein, for example, changes in the substrate in the active center
of the enzyme. There arc ligands that can attach, to the protein only at the moment of
functioning, for example, O., transported by hemoglobin. Ligands can be constantly
associated with the protein, which plays an auxiliary role in the functioning of proteins,
for example, iron which is part of hemoglobin. The connection of protomers in an
oligomeric protein is an example of the interaction of high molecular weight ligands.
Each protomer connected lo other protomers serves as a ligand for them, just as they
do for it. Sometimes the attachment of a ligand changes the conformation of the
protein, resulting in the formation ofa binding center with other ligands. Tor example,
the protein calmodulin, after binding to four Ca2 ions in specific areas, acquires the
ability to interact with some enzymes, changing their activity.

Protein classification
Protein classification is based on chemical composition, structure, functions,
and solubility in different solvents. Based on their chemical composition, proteins
may be divided into two classes: simple and complex. Simple proteins also known
as homop rote ins, they arc made up of only amino acids. For examples, arc plasma
albumin, collagen, and keratin. Complexprotc ins sometimes also called hctcroprotcins,
except amino acids, have non-protcin components. The non-protcin part is called the
prosthetic group, and the protein part — apoprotein. Complex protein — holoprotein —
can dissociate into components:

holoprotein < > apoprotein 4- prosthetic group

The direction of the reaction depends on the bond strength of the holoprotcin
components. The prosthetic group can. be organic substances, metal ions, nucleic
acids, carbohydrates, lipids and other substances (Table 1.2).
Table 12. Complex protein examples

Metal Proteins Metal Ions

Phosph op roteins H3POt

Hemo prate ins Hemes

Glycoproteins Monosaccharides, o ligosaccharid es

Flavoproteins Flavinnucleotides

Proteoglycans Glycosaminoglycans

Lipoproteins Triacylglycerols and Complex Lipids

Nucleoproteins:
ribo nucleoproteins (ribosomes, etc.); RNA
deoxyribonuefeoproteins ; chromatin i DMA
1.3. Trie Levels of Protein Structures: Primary, Secondary, Tertiary 29

Glycoproteins arc proteins that covalently bind one or more carbohydrate units
to the polypeptide backbone. Typically, the branches consist of not more than 15-
20 carbohydrate units. Examples of glycoproteins arc: glvcophorin, the best known
among erythrocyte membrane glycoproteins; fibronectin, that anchors cells to the
extracellular matrix through interactions on one side with collagen or other fibrous
proteins, while on the other side with cell membranes; all blood plasma proteins,
except albumin: immunoglobulins or antibodies. Phosphoprotcins are proteins
that bind phosphoric acid to serine and threonine residues. Generally, they have a
structural function, such as tooth den Lin, or reserve function, such as milk caseins
( alpha, beta, gamma and delta), and egg yolk phosvitin.
Based on their shape, proteins may be divided into two classes: fibrous and globular.
Fibrous proteins have primarily mechanical and structural functions, providing
support to the cells as well as the whole organism. These proteins arc insoluble in water
as they contain, internally as well as on their surface, many hydrophobic amino acids.
The presence on their surface of hydrophobic amino acids facilitates their packaging
into very complex supra molecular structures. In this regard, it should be noted that
their polypeptide chains form long filaments or sheets, where in most cases only one
type of secondary structure, that repeats itself, is found.
Here arc some examples, collagen which constitutes the main protein component
of connective tissue, and more generally, the extracellular scaffolding of multicellular
organisms, a-Ke rati ns constitute almost the entire dry weight of nails, claws, beak,
hooves, horns, hair, wool, and a large part of the outer layer of the skin. Most of the
proteins belong to the class of globular proteins.
They have a compact and more or less spherical structure, more complex than
fibrous proteins. In this regard, motifs, domains, tertiary and quaternary structures
arc found, in addition to the secondary structures. They arc generally soluble in water
but can also be found inserted into biological membranes (transmembrane proteins),
thus in a hydrophobic environment. Unlike fibrous proteins, that have structural and
mechanical functions, they act as:
* enzymes:
* hormones;
► membrane transporters and receptors:
» transporters of triglycerides, fatly acids and oxygen in the blood:
► immunoglobulins or antibodies;

Substances that affect the functioning of the protein

Ligands interact with proteins and arc able to change their conformation in such
a way that new active centers arc formed for communication with other ligands.
As mentioned above, the interaction, of a protein with a ligand has high specificity.
Nevertheless, it is always possible to choose a substance (natural or synthetic), which
is a structural analog of the ligand and can be complementary to the active center of
the protein. If this substance binds to a protein instead of a natural ligand, protein
function will be blocked. A substance that interacts with the active center of a protein
and blocks the action of a protein is called a protein inhibitor or antagonist. If this
substance is similar in structure to a ligand, it is called a structural analog of the
30 Cnaptef 1. Tn e structure, properties and functions of proteins

ligand. It also interacts with the active center of a protein. An analog that replaces
a natural ligand in an active center protein and reduces its function is called a
competitive protein inhibitor
Analogs of natural protein ligands arc used as medicines. Such drugs a re widely used
in the regulation of transmission of excitation through synapses. The neurotransmitter
secreted during the passage of the pulse by the nerve endings should interact highly
with receptor proteins on the postsynaptic membrane. However, by modifying the
chemical structure of the neurotransmitter, it is possible to obtain substances that
would also bind to the receptor, but the physiological effect changed, decreased or
intensified. In pharmacology; such substances arc called *antagonisls*and ^agonists*,
respectively.
Dilhilin (a pharmacological substance) is an analog of acetylcholine, which
ensures the transmission of a nerve impulse to a muscle, being a synapse mediator.
When dilhilin is introduced into the body; it binds to the N-cholinergic receptors
of cholinergic synapses, being an antagonist of acetylcholine, a neurotransmitter
that causes muscle contraction. As a result, the transmission is blocked and muscle
relaxation (paralysis) occurs. Therefore, dilhilin is used as a muscle relaxant in.
operations and endoscopic examinations. Another drug that acts as a protein inhibitor
is atropine, an alkaloid of plant origin, which is also a natural analog of acetylcholine,
but interacts with M-cholinergic receptors of the membrane of effector cells. Being an
acetylcholine antagonist, it eliminates the irritation of parasympathetic nerves, and
instead of smooth muscle contraction, which, is stimulated by acetylcholine, relieves
their spasm.
Some poisons, getting into the human body, firmly bind to certain proteins,
inhibit them and thereby cause disturbances in biological Junctions. For example,
cobra neurotoxins specifically interact with, cholinergic receptors of postsynaptic
membranes, blocking their work. The affinity of neurotoxins for cholinergic receptors
causes to the formation of many bonds between the toxin and the receptor, which
leads to their practically irreversible connection. It must be remembered that there is
often a transparent border between drugs and poisons, and the effect of their action
depends on the dose of the substance administered. Thus, drugs prescribed in doses
greater than therapeutic can act as poisons, i.e. cause serious metabolic disorders
and disruptions of body functions, and poisons in microdoscs arc often used as
medications. For example, atropine, which is widely used to relieve spasms of smooth,
muscles, in high doses causes CNS excitement, and in even higher doses, it causes
a sleep that goes into a coma. Known hypotensive agent clonidinc in the case of an
overdose causes collapse.
1.3. The Levels of Protein Structures: Primary, Secondary, Tertiary

Review tests
I. Choose one correct answer. The peptide contains:
H o H C
l I !

COOH

A. Three peptide bonds.

B. Lysine with a free a-amino group.
C. Tyrosine with a free a-carboxyl group.
D. Only polar radicals.
E. Tyrosine with a free a-amino group.
2. Choose one correct answer. The primary structure of the protein:
A. Is formed by hydrogen bonds between adjacent amino acids.
B. Is destroyed at extreme pH values.
C. Encoded in a DNA molecule.
D. Depends on the type of cells synthesizing it.
E. Capable of denaturation.
J. Which one of the rollowing bonds is involved in the formation of the secondary structure
of the protein:
A. Peptide.
B. Hydrogen.
C. Disulfide.
D. Ionic.
E. Hydrophobic.
4. Choose one correct answer. A peptide containing only hydrophobic amino acid
radicals:
A. Val-Pro-Thr-Trp.
B. llc-Leu-Trp-Ala.
C. M ct -Se r- Leu -A rg.
D. Pro-Tyr-Val-Gly.
E. Ik-Trp-Gln-Mel.
5. Denaturation and renaturalion of ribonuclease. Match the figure w ith the letter:
32 Chapter 1. Tnb structure, properties and functions of proteins

A. Renailiration.
B. Disulfide bonds.
C. Native protein.
D. Denaturation.
E. Cysteine radicals.
6. Match the figure with the Idler Secondary struct urc of protein:

A. N-terminal end.
B. Parallel p-sheet.
C. C-terminal end.
D. Antiparalfcl p-shecl.
E. Hydrogen bonds between the atoms of the peptide backbone.
7. Match the figure with the letter. Levels of structural organization of proteins:
1. The primary structure.
2. The secondary structure.
3. Tertiary structure:
a) spatial folding of the polypeptide chain:
b) the order of alternation of amino acids:
c) a structure formed by imcrradical interactions:
d) spatial stacking of the peptide backbone:
e) the specific arrangement of secondary structures.
8. Match the figure with the letter. Protein-ligand interaction:

A. ProLein.
B. A ligand.
C. Peptide bonds.
D. Active center.
E. Weak connections.
1.4. Quaternary Structure of Proteins. Hemoglobin 33

Situational Problems
1. The use of natural proteins as drugs requires compliance with certain storage
and use conditions, which arc clearly prescribed in. the instructions. So, most
protein preparations must be stored in a refrigerator al a temperature not excee­
ding 10 ’C3 and dry prepara Lions should be dissolved in boiled water cooled to
room temperalLire. Some protein preparations are stored in hermetically scaled
ampoules, from which air is removed. Why do protein drugs require strict
adherence to all the conditions prescribed in the instructions, and what can
happen if these conditions arc violated?
2. Atropine is an alkaloid found in some plants: belladonna, dope, and bleach. Il is
pan. of the drugs belonging to the group of antispasmodics that relieve smooth
muscle spasms. Such, drugs are used for spastic pain and stomach, intestines, to
relax the smooth, muscles of the bile ducts. What is the mechanism of action of
this substance?

14. QUATERNARY STRUCTURE OF PROTEINS. HEMOGLOBIN

If proteins consist of two or more polypeptide chains linked by non-covalent bonds,
then they arc said to have a quaternary structure. Such aggregates arc stabilized by
hydrogen bonds, ionic bonds and electrostatic interactions between oxygen residues
located on the surface of the globules. Such proteins arc called oligomers, and their
individual chains arc called protomers (monomers, subunits). Proteins with identical
subunits arc termed homo-oligomers. Proteins containing several distinct polypeptide
chains arc termed hetero-oiigomers.
The formation of proteins with a quaternary structural organization allows
organism to expand its capabilities in the field of qualitative diversity of proteins
with a slight increase in the quantity of the genetic material. For example, the
lactate dehydrogenase (LDH) enzyme, consisting of 4 subunits, is formed from
2 genetically determined polypeptide chains H and M. Their different combinations
(HHHH, HHHM, HHMM. HMMM, MM MM) allow 5 LDH enzymes to exist in
the body, catalyzing the same reaction in different organsand tissues: LDH1, LDH2,
LDH3, LDH4 and LDH5. Such proteins with identical functions, but differing in
physicochemical properties, arc called isoproteins.
Oligomeric proteins exhibit properties absent in monomeric proteins. The
influence of t he quaternary structure on the functional properties of the protein can be
considered by comparing the structure and functions of two related heme containing
proteins: myoglobin and hemoglobin. Both proteins have a common evolutionary
origin, a similar conformation of individual polypeptide chains and a similar function
(participate in oxygen transport), but myoglobin is a monomeric protein and
hemoglobin is a tetramer. The presence of a quaternary structure in hemoglobin gives
this protein properties that arc absent in myoglobin.

Myoglobin
Myoglobin is classified as he me-containing proteins, i.e. it contains a prosthetic
group - heme, quite firmly connected with the protein part. Myoglobin is classified
34 Cnaptef 1. Tn e structure, properties and functions of proteins

as a globular protein; it has only one polypeptide chain. Myoglobin is found in red
muscles and is involved in oxygen storage. In conditions of intense muscular work,
when the partial pressure of oxygen in the tissue drops, O2 is released from the complex
with myoglobin and is used in the mitochondria of cells to obtain the energy necessary
for muscle work.
Heme is a molecule with the structure of cyclic tctrapyrrolc, where 4 pyrrole
rings are connected by methylene bridges. This organic part of the heme is called
protoporphyrin.
In heme. 4 nitrogen atoms of the pyrrole rings of protoporphyrin IX arc linked by
four coordination bonds with Fe2‘ located in the center of the molecule {Fig. 1.18).

Fig. 11tL Myoglobin structure. Heme is a complex of porphyrin and ferrous iron (IV-

Apomyoglobin — the protein part of myoglobin; the primary structure is

represented by a sequence of 153 amino acids, which arc arranged in 8 a-helices in the
secondary structure. The a-heliccs arc denoted in Latin letters from A to H. starting,
from the N-terminus of the polypeptide chain. The tertiary structure has the form
of a compact globule (there is practically no free space inside), formed due to loops
and turns in the region of non-helical sections of the protein. The inner part of the
molecule consists almost entirely of hydrophobic rad icals, with the exception of two
histidine residues located in the active center.
Heme is a specific apomyoglobin ligand that joins the protein part in the pocket
between the two a-helices F and E. The heme binding site is formed mainly by
hydrophobic amino acid residues surrounding the hydrophobic pyrrole rings of the
heme.
In addition to hydrophobic amino acids, the active center of apomyoglobin also
includes 2 histidine residues (His E7 and His F8), which play an important role in
the functioning of the protein. They arc located on different sides of the heme plane
and are part of the F and E helices, between which the heme is located. The iron
atom in the heme can form 6 coord ination bonds, 4 of which hold Fc2' in the center
of protoporphyrin (connecting it with the nitrogen atoms of the pyrrole rings), and
the 5th bond arises between Fe2’ and the nitrogen atom of the imidazole ring His F8
(Fig. I t?).
1.4. Quaternary Structure of Proteins. Hemoglobin 35

His E7, although not associated with he me, is necessary for the proper orientation
and attachment of another ligand O2 to myoglobin. The amino acid environment of
the heme creates the conditions fora rather strong, but reversible binding of O, to Fc2'
myoglobin. The hydrophobic amino acid residues surrounding heme prevent water
from entering the myoglobin, binding site and the oxidation of Fc3, to Fc3\. Trivalent
iron in the heme is not able to attach oxygen.

Fia 119- The location of the heme in me active center ol apomioglehin and he mo globin protomers

Hemoglobin
Hemoglobins are related proteins found in. human and vertebrate erythrocytes.
They are also classified as heme-containing proteins. These proteins perform
2 important functions:
► transfer of oxygen from the lungs to the peripheral tissues;
► participation in the transfer of CO., and protons from peripheral tissues to the
lungs for subsequent excretion from the body.
The most important characteristic of hemoglobin is its ability to regulate the
affinity for O3depending on tissue conditions. Hemoglobins arc similar in structure
to myoglobin, but they have a quaternary structure (they consist of 4 polypeptide
chains), which makes it possible to regulate their functions. In adult red blood cells,
hemoglobin accounts for 90% of all proteins in a given cell.
Adult Hemoglobins:
► hemoglobin A (HbA) the main adult hemoglobin, accounts for about 98% of the
total hemoglobin, tetramer, consists of 2 polypeptide chains a and 2p (2a2p):
* hemoglobin A2 is in the adult body in a lower concentration, it accounts for about
2% of total hemoglobin. It consists of 2a and 20 chains (2a2O);
* hemoglobin Ale is hemoglobin A modified by the covalent addition of glucose to
it (the so-called glycosylated hemoglobin).
Hemoglobins synthesized during fetal development:
► embryonic hemoglobin (HbE) is synthesized in the fetal yolk sac a few weeks
after fertilization. h is a tetramer 2g2e. After 2 weeks of formation of the fetal
36 Chapter 1. Tnb structure, properties and functions of proteins

liver., lie moglobin F begins to be synthesized in it, which, at 6 months replaces

embryonic hemoglobin;
► hemoglobin F (HbF) isa fetal hemoglobin that is synthesized in the liver and bone
marrow of the fetus before its birth. It hasa tetra meric structure consisting of 2a
and 2y chains. After the birth of a child Jt is gradually replaced by hemoglobin A.
The conformation of individual hemoglobin protomers is very similar to the
conformation of myoglobin, despite the fact that only 24 amino acid residues arc
identical in the primary structure of their polypeptide chains. The hemoglobin
protomers, like apomioglobin, consist of 8 helices folded into a dense globular
structure containing an internal hydrophobic core and a ^pocket* for heme binding.
The heme compound with globin (protein part) is similar to that of myoglobin — the
hydrophobic environment of the heme, with the exception of 2 residues His 7 and
His FS (Fig. 1.18, 1.19). However, the tetrameric structure of hemoglobin isa more
complex structural and functional complex than myoglobin.

The role of histidine E7 in functioning of myoglobin and

hemoglobin
Heme hasa high affinity for carbon monoxide (CO). In the aquatic environment,
the protein-free heme binds to CO 25,000 times stronger than 0.,. The high degree of
heme affinity for CO compared to O is explained by the different spatial arrangements
of the Fe2' heme complexes with CO and O2. In the Fe2' heme complex with CO, the
Fe2', carbon, and oxygen atoms arc located on one straight line, and: in. the Fe2 heme
complex with the iron and oxygen atoms arc located at an angle, which re lice ts
their optimal spatial arrangement.
In myoglobin, and hemoglobin above Fe2* in the region of attachment O., His E7 is
located, which disrupts the optimal arrangement of CO in the center of protein binding
and weakens its interaction wiih heme. On the contrary, the same His E7 creates
optimal conditions for the binding ofO (Fig. 1.19). As a result, the hemo affinity to
CO in proteins is only 200 limes higher than its affinity for O,. Decreased affinity for
heme-containing proteins to CO is of great biological importance. CO is produced in
small amounts during the catabolism of certain substances, in particular heme. This
endogenously formed CO blocks about 1% of heme-containing proteins. If the heme
affinity for CO did not decrease under the influence of the protein environment, then
endogenous carbon monoxide could cause serious poisoning.

Quaternary hemoglobin structure

Four hemoglobin polypeptide chains connected together form an almost regular
ball shape, where each a-chain is in contact with two tJ-chains (Fig. 1.20.). There
arc many hydrophobic radicals in the contact region between al and pl, and also
between a2- and 32-chains. Therefore, a strong compound is formed between these
polypeptide chains lor hydrophobic as well as ionic and hydrogen bonds. As a result,
dimers a 131 and a2p2 are formed.
Polar (ionic and hydrogen) bonds arise between these dimers in the tetrameric
hemoglobin molecule; therefore, when the pH of the medium changes io the acidic
1.4. Quaternary Structure of Proteins. Hemoglobin 37

or alkaline side. the bonds between the dimers arc first destroyed. In addition, dimers
arc able to easily move relative to each other. Since the surface of the protomers is
uneven, the polypeptide chains in the central region cannot adhere closely to each
other; as a result, a ^central cavity* is formed in the center, which passes through
the entire hemoglobin molecule. The weaker interactions between dimers result in
the two dimers occupying different relative positions in deoxyhemoglobin (T form) as
compared with oxyhemoglobin (R form) (Fig. 1.21).

Heme

Heme

fl - polypeptide ci - polypeptide
(globin) chain (globin) chain

Fig. 120. Quaternary structure of hemoglobin A

Weak. ionic and Sirong meractcrrs, Sot ■? tonic- and

bydragan bonds occur primady hydraptibbic. hydrogen bonds
beween u> efimer pairs bKween a and |i ceiweon up dimers
in ihe deoxygenated siare ttians farm statte are broken in ihe
tifi amers. oxygenawd stare.

r
4O2

T7 or tam, suuciure of decxyfiemcgicthn ■R," or relaxed, svuciure of oxyfiemcglothn

Fig. 121. interaction between hemoglobin subunils

O2 binds to hemoglobin protomers via Fe2‘, which is connected to four nitrogen

atoms of the hemo pyrrole rings and His F8 nitrogen atom. Oxygen is bound to the
remaining free coordination bond Fc21 on the other side of the home plane in the His
E7 region. In deoxyhemoglobin, the Fc2‘ atom, due to the covalent bond with the
38 Chapter 1. Tnb structure, properties and functions of proteins

protein pan, protrudes from the heme plane in the direction of His F8. The addition
of O2 to the Fc2- ion of one protomer causes it to move to the heme plane; the His
F8 residue and the polypeptide chain of which it enters also move behind it. Since
proteins have conformational lability, the conformation of the whole protein changes.
Conformational changes that occurred in other protomers facilitate the attachment of
the next O2 molecule, which, causes new conformational changes in the protein and
accelerates the binding of the next ()., molecule. The fourth ()., molecule attaches to
hemoglobin 300 times easier than the first molecule (Fig. 1.22).

\ /H \

. . . C— N . . . C—N
Histidine a Histidine

tZ \A
Fe

Heme is domed
(nonplanar)

Deoxygenated Oxygenated

Increasing
affinity

Fig. 122. Cooperative changes in the conformation of hemogl oh in protomers upon addition of 0,
1.4. Quaternary Structure of Proteins. Hemoglobin 39

A change in the conformation and, as a consequence, the functional properties of

all. protomers of the oligomeric protein upon ligand attachment to only one of them
is called cooperative changes in the con format ion of protomers. Similarly, in tissues
the dissociation of each O, molecule changes the conformation of all protomers and
facilitates the cleavage of the subsequent ()., molecules.
The cooperative effect of hemoglobin protomers can also be observed on the
()-, dissociation curves for myoglobin and hemoglobin (Fig. 1.23). The ratio of O-,
occupied protein binding sites to the total number of such binding sites is called the
degree of oxygen saturation of these proteins. Dissociation curves show how saturated
these O2 proteins arc for various values of the partial oxygen pressure.

The oxygen dissociation curve for Hb is steepest

at the oxygen concentrations that occur in the tissues.
This permits oxygen delivery to respond to small
changes in pO2-

pO2 in pO£ in

Fig. 1-23.Oxygen tfissociatiou curves for myoglobin and hemoglobin depending on the partial pressure
of oxygen

Myoglobin binds oxygen, which releases hemoglobin in the tissue capillaries, and
myoglobin itself can release OJn response to increased demand for muscle tissue and
intensive use of 07 during exorcise. Myoglobin has a very high affinity forO.,. Even al
a partial pressure ofO,, equal to 1.-2 mm Hg myoglobin remains bound to O by 50%.
Hemoglobin has a significantly lower affinity for O:,; half-saturation of hemoglobin
occurs at a higher pressure of O3 (about 26 mm Hg). The hemoglobin dissociation
curve has a sigmoid shape (S-shaped). This indicates that the hemoglobin protomers
work cooperatively: the more is released by the protomers, the easier is the cleavage
of subsequent O2 molecules.
40 Cnaptef 1. Tn e structure, properties and functions of proteins

Due to the unique structure, each of the considered proteins is adapted to perform
its function: myoglobin — attach O2 released by hemoglobin, accumulate it in. the coll
and give it up, if necessary. Hemoglobin is an additive in the lungs to O2, where its
saturation reaches 100%, and release O, in die capillaries of tissues, depending on. the
change in O2 pressure, in them.

Transfer of H* and CO2 from tissues to the Lungs by hemoglobin.

Bohr effect
In the mitochondria of cells, organic substances arc oxidized with oxygen to
produce energy. In this case, the final decomposition products CO3 and 1-1,0 are
formed, the amount of which is proportional to the intensity of oxidation processes.
CO. formed in the tissues is transported to red blood celts. There, under the action
of the enzyme carbonic anhydrase, which turns it into carbonic acid, an increase in
the rate of its formation occurs. Weak carbonic acid H2COj can dissociate into H ‘
and HCOj . In erythrocytes located in the tissue capillaries, the reaction equilibrium
shifts to the right, since protons formed as a result of dissociation of carbonic acid can
attach to specific parts of the hemoglobin molecule: His46 radicals of two (3-chains,
Hisl22 radicals and terminal a-amino groups of twoa-chains. All these 6 sites, upon
the transition of hemoglobin from oxy to deoxy form, acquire a greater affinity for
H as a result of a local change in the amino acid environment around these sites
(the approach of negatively charged carboxy] groups of amino acids). The addition of
3 pairs of protons to hemoglobin reduces its affinity for O2 and enhances the transport
ofO, to tissues that need it (Pig. 1.24). The increase in the release of O, hemoglobin
depending on the concentration of H is called the llohr cfleet, after the Danish
physiologist Christian Bohr, who first discovered this effect.

The Bohr Effect

pH 7.2 — periphery pH 7.4

x
pH 7.6 — lung
■o 1004 higher affinity,
CD O.? load
A) 80-
CD
tn 60-
CD Lower affinity;
40- O2 unloading

20-■
0 20 40 60 SO 100
Po2(mmHg)

Fig. 124. Hemoglobiu-oxyge11 dissociation! curve and Bohr effect

1.4. Quaternary Structure of Proteins. Hemoglobin 41

In the lung capillaries. the high partial pressure of O] leads to oxygenation of the
hemoglobin and removal of 6 protons. The reaction CO2+ + HCO2
is shifted to the left and the formed CO2 is released into the alveolar space and is
removed with exhaled air (Pig. 1.25). During evolution, the hemoglobin molecule
acquired the ability to perceive and respond to environmental changes. An increase in
the concentration of protons in the medium decreases the affinity ofO3 for hemoglobin
and enhances its transport into tissues.
Most CO2 is transported by blood in the form of HCO, bicarbonate. A small
amount of CO (about 15-20%) can be transferred to the lungs, reversibly attaching
to non-ionized terminal ot-amino groups. As a result, carboxyhemoglobin is formed.
The addition of CO2 to hemoglobin also reduces its affinity for Or

CO2 is released O2 binds to

from hemoglobin hemoglobin

Fig. 125. Transfer of and C0s with hlood

42 Chapter 1. Tnb structure, properties and functions of proteins

2r3-bisphosphuglycerate is an allosteric regulator of hemoglobin

affinity for tX
23-bisphosph.oglyccraLe (2,3-HPG) is a substance synthesized in red blood
cells from an intermediate product of 1,3-bisphosphoglyccratc as a result of glucose
oxidation. Under normal conditions. 2,3-bisphosphoglyceraic is present in red
blood cells at approximately the same concentration as hemoglobin. 2,3- BPG, when
combined with hemoglobin, can also change its affinity for O2
Al the center of the tetrameric hemoglobin molecule, there is a cavity formed by
amino acid residues of al l four protomers. The central cavity is the site of attachment
of 2,3-BPG. The dimensions of the central cavity can. vary; the removal of O2 from
oxyhemoglobin causes its conformational changes, which contribute to t he formation
of additional ionic bonds between dimers al pl and o2p2. As a result, the spatial
structure of deoxyhemoglobin becomes more tense and the central cavity expands
(Fig I.26).
Salt bridges
| 2,3-BPG

>.—J V '* ‘J

Hz'dz |‘ ci ■

Oxyhemoglobin | Deojcyhemoqlobin I

«.,4
$A
sutjunrt

Vj

oo
p2 subunh *

Lys92

His 2

His 143

Fig. 126.1iHenachon of 2J-hisphosphoglycerate with hemoglobin

In the central cavity; there arc positively charged radicals of amino acid residues
and positively charged a-amino groups of the N-terminal partofp-chains. 2,3-BPG,
which has a strong negative charge, is attached to the expanded deoxyhemoglobin
1.4. Quaternary Structure of Proteins. Hemoglobin 43

cavity by means of ionic bonds formed with positively charged functional groups. The
addition of 2,3-B PG even more stabilizes the taut form structure of deoxyhe moglobin
and reduces its affinity for O2. The addition of2,3-BPG to dcoxyhcmoglobin occurs in
a different site, compared with the active site where O2 binding occurs. Such a ligand
is called allosteric and the center where the allosteric ligand is bonded is the allosteric
center («allos* — is another, different, micros* — is spatial). In the lungs, a high partial
pressure of O, leads to hemoglobin oxygenation. The rupture of ionic bonds between
dimers al pl and a2p2 leads to relaxation of the protein molecule, a decrease in the
central cavity and the displacement of 2,3-BPG.
The concentration of 2,3-BPG in the erythrocytes of people living in certain
climatic conditions is a constant value. However, in the period of adaptation to high
mountains, when a person rises to an altitude of more than 4000 m above sea level,
the concentration of 2,3-BPG in almost 2 days increases almost twice (from 4.5 to
7.0 mM). This reduces the affinity of hemoglobin for O3 and increases the amount of
O., transported to the tissue (Fig. 1.27) Changing the concentration of 2,3-BPG works
as a mechanism for adapting the body to hypoxia. The same adaptation is observed in
patients with lung diseases, in which general tissue hypoxia develops.

2,3-BPG - flinnxH/L (Blood 1-rorn mc&vsduSadapiM id Tpgh ahiiudes}

[ 2.3-BPG - mimoL.t ^HenwglotSn cl 2,3 B~'G;

Partial pressure ol oxygen (pO?)

(mm Hg)

Fig. 127. Allosteric effect of 2,3-BPG on the oxygen affinity of hemoglobin

Fetal hemoglobin (HbF) replaces embryonic hemoglobin (HbE) after 2 weeks

formation of the fetus liver. From 6 months of fetal development until birth, it is the
main hemoglobin of red blood cells. After the birth, of a child, it begins to be intensively
replaced by hemoglobin A (HbA). Under physiological conditions, HbF has a higher
affinity for O than HbA. which creates optimal conditions for the transport of O
from maternal blood to fetal blood. This property of HbF is due to the fact that it is
weaker than HbA binds to 2,3-BPG. The physiological features of HbF arc related to
the peculiarities of its structure: instead of p-globin chains in HbA. it contains two
44 Chapter 1. Tnb structure, properties and functions of proteins

Y-chains. Some of the positively charged amino acid radicals arc absent in the primary
struct tire of the y chains. In a medium lacking 2,3-11 PG, HbA and HbF exhibit the
same high affinity forO3.

Hereditary hemoglobinopathy
The importance of the primary structure of proteins for the formation of their
conformation and function can be Lraccd to the examples of hereditary diseases
associated with a change in the primary structure of hemoglobin. At present, about
.300 HbA variants arc known that have only small changes in the primary structure of
the a or p chai ns. Some of them ba rely affect protein function and human health, others
reduce protein function, and especially in extreme situations, reduce the possibility
of human adaptation, others cause significant impairment of HbA. functions and the
development of anemia, which leads to serious clinical consequences.
In 1904, a Chicago doctor, J. Herrick, described severe anemia in a student with
the discovery of many elongated, sickle-1 ike red blood cells in his blood. The disease
was called sickle cell anemia, and only in 1949 did L. Pauling and his colleagues prove
that it was caused by a change in the primary structure of HbA. in the hemoglobin
S (HbS) molecule (the so-called abnormal hemoglobin), 2 P-chains arc mutant, in
which glutamate, the highly polar negatively charged amino acid at 6,k position, was
replaced by a valine containing a hydrophobic radical (Fig. 1.28).

NORMAL SICKLE CELL

GAG MUTATION GTG

DNA
CT C ■ » CAC
J—L J—L
I 1
1—
RIMA
GAG GuG
1 1
-.ULLf □MX
PROTEIN
NORMAL MUTANT
PROTEIN PROTEIN

Fig. 1.28. Hemoglobin S (HbS) is a mutant Iona of hemoglobin

In deoxyhemoglobin S. there is a site complementary io another site of the same

molecules containing an altered amino acid. As a result, deoxyhemogtobin molecules
begin to stick together, forming elongated fibrillar aggregates that deform red blood
cells and lead to the formation of abnormal red blood cells in the form of a. sickle.
In oxyhemoglobin S, the complementary region is « masked* as a result of changes
in protein conformation. The inaccessibility of the site prevents the connection
of oxyhemoglobin S molecules with each other. Consequently, the formation
of HbS aggregates is facilitated by conditions that increase the concentration of
deoxyhemog lobin in cells (physical work, hypoxia, decrease in pH, high, altitude
conditions, etc). Since sickle-shaped erythrocytes do not pass well through tissue
capillaries, they often clog vessels and thereby create local hypoxia. Disruption of O3
1.4. Quaternary Structure of Proteins. Hemoglobin 45

delivery io tissues causes pain and even cell necrosis in. this area. Sickle cell anemia is a
homozygous recessive disease; it only manifests itself in the case when 2 mutant genes
p-chains of globin arc followed from both parents (Fig. 1.29).

When both parents When one parent has Sickle Cell

have Sickle Cell & anotner has the Sickle Cell Trait

H M Sickle Ceil Sickle C all Trait Sickle Cal I

it ii it it Sickle Cell

Risk for child Id:

Sickle Cell Trait

Risk for child Id:

Sickle Cell

Have Sickte Cefl 100% Have Sickte Cell 50%

Have Sickte Cefl Trait 0% Have Sickte Cell Trait 50%
h______________________________________ < i______________________________________ j

When both parents have When one parent has

the Sickle Cell Trait the Sickle Cell Trait

H H Sickle Cell Trail; Sickle Cell Trait unaffected

il #1 ♦t li
Unaffected [Sickle Cell Trait] Sickle Cell

Risk for child Id:

unaffected

Risk for child Id:

Sickte Cell Trait

Have Sickte Cell 25% Have Sickte Cefl 0%

Have Sickte Ceffl Tract 50% Have Sickte Ceti Trait 50%

Fig. 129 inheritance of sickle cell anemia and manifestation of the disease

After the birth of a baby, the disease docs not appear until significant amounts
of HbF arc replaced: by HbS. Patients reveal clinical symptoms characteristic for
anemia: dizziness and headaches, shortness of breath, palpitations, pain in the limbs.
46 Chapter 1. Tnb structure, properties and functions of proteins

increased susceptibility to infectious diseases. Heterozygous individuals having one

normal HLA gene and another HbS gene in blood have only traces of sickle cells and
a normal lifespan: clinical symptoms of the disease usually do not appear.
Another example of hemoglobinopathy is methemoglobin. Hemoglobin M is
a variant of hemoglobin A, where the presence of a mutation in the a- or fl-chain
gene, the His E7 or His F8 is replaced by tyrosine. As a result of such mutation, Fc2'
is oxidized to Fc1' and stabilizes in this form. The hemoglobin containing Fc1' in
the hemo is called methemoglobin (hemoglobin M). Instead of O H,O is attached
to Fcv. Usually, changes affect either the a- or P-chains. so no more than two O3
molecules can be carried by hemoglobin. In heterozygous people, cyanosis associated
with impaired transport is noted; and homozygosity for this gene leads to death.
There arc several hereditary hemoglobinopathies that most often lead to impaired
oxygen transport as well as to hypoxia or cyanosis.

1.5. PHYSICAL CHEMICAL PROPERTIES OF PROTEINS

Individual proteins differ in their physicochemical properties: the shape of the
molecules, molecular weight, total charge, solubility, etc.

Differences in the shape of molecules

As mentioned above the form of protein molecules is divided into globular and
fibrillar. Globular proteins have a more compact structure, their hydrophobic radicals
arc mostly hidden in a hydrophobic core, and they arc much better soluble in body
lluids than fibrillar proteins (the exception is membrane proteins).

Molecular weight differences

Proteins arc high-molecular compounds, but can vary greatly in molecular weight,
which ranges from 6,000 to 1,000,000 I) and higher. The molecular weight of the
protein depends on the number of amino acid residues in the polypeptide chain, and for
oligomeric proteins it depends on the number of protomers (or subunits) included in it.

Total charge of protein

Proteins have the property of amp hole ricity. That is, depending on the conditions,
they exhibit both acidic and basic properties. In proteins, there arc several types of
chemical groups capable of ionization in an aqueous solution: carboxylic acid residues
of the side chains of acidic amino acids (aspartic and glutamic acids) and nitrogen-
containing groups of the side chains of basic amino acids (primarily the amino group
of lysine and the amid residue CNH (NH,) of arginine).
Protein charge depends on the ratio of acidic and basic amino acids. Therefore,
like amino acids, proteins charge positively with decreasing pH and negatively
with increasing it. If the pH of the solution corresponds to the isoelectric point of
the protein, then the charge of the protein is 0. If acidic amino acids {glutamate
and aspartate) predominate in the peptide or protein, then the protein is acidic, at
neutral pH the protein charge is negative and the isoelectric point is in the acidic
environment. For most natural proteins, the isoelectric point is in the pH range of
1.5. Physical Chemical Properties of Proteins 47

4.S-5.4. which indicates the predominance of glutamine and aspartic amino acids
in their composition. If the protein is dominated by basic amino acids (lysine and
arginine), then al neutral pH the protein charge is positive and it is due io these
positively charged amino acids.
Amphocericity is important for proteins to perform certain functions. For example,
the buffer properties of proteins? i.e the ability to maintain unchanged blood pH .
based on the ability io attach H ions during acidification of the environment or give
them when alkalizing. On the practical side, the presence of amphotericity makes it
possible to separate proteins by charge (electrophoresis) or use a change in the pH
of the solution to precipitate any known protein. The presence of both positive and
negative charges in a protein determines their ability to salting out, which is convenient
for isolating proteins in a native (living) conformation.
As the pH in the solution changes, the concentration of H ions changes loo.
When the medium is acidified (with a decrease in pH) below the isoelectric point,
H ions attach to the negatively charged groups of glutamic and aspartic acids and
neutralize them. The protein charge in this case becomes positive. With increasing pH
in the solution above the isoelectric point, the concentration of H ions decreases and
positively charged protein groups (N H, groups of lysine and arginine ► lose protons,
their charge disappears. The total charge of the protein becomes negative 4 Fig. 1.30).

+H* +c+r
NH.— Ch— COOH * - nhZ— Ch — COO" * ■ Ch — COO"
nonpolar and I
polar radicals H H Fl
| net charge = +l 1 | ne* charge = 0 | net charge =-■!

+H* 1-CH"
NHg‘— CH — COOH - - nhZ—Ch — COO" * NFk—CH — COO-
1 |i |
- charge CH.
radicals I
COOH COCr COO"
[ net charge = +l netchajge=-l i | net charge =-’)

t+T tOH"
NH3 Ch COOh * NH3 CH COO" * nh2— Ch—■COG-

+ charge (CH^h (CHah (Wc

1 ♦
radkals 1 .
MH/
I
NHz

| net charge =+2 j net charge =+1 | net charge

Fig. 1.30. Changes in the total charge at a pratein with a change in pH

The pH value al which the protein acquires a total zero charge is called the
isoelectric point and is denoted as pl. At the isoelectric point, the number of positively
and negative ly charged protein groups is the sameT i.e. the protein is in an isoelectric
state. Since most of the proteins in the cell contain more anionic groups (-COO-),
48 Cnaptef 1. Tn e structure, properties and functions of proteins

the isoelectric point of these proteins lies in a slightly acidic medium. The isoelectric
point of the proteins, in which cat io nogen ic groups predominate, is in an alkaline
environment. The most striking example of such intracellular proteins containing a lot
of arginine and lysine arc histones, which arc part of chromatin.
Proteins having a total positive or negative charge arc better soluble than proteins
located al an isoelectric point. The total charge increases the number of water dipoles
that can bind to a protein molecule, and prevents the contact of the same charged
molecules, as a result, the solubility of proteins increases. Charged proteins can move
in an electric field: anionic proteins having a negative charge will move to a positively
charged anode (+), and cationic proteins to a negati vely charged cathode (-). Proteins
in an isoelectric state do not move in anelectric field. At the isoelectric point, proteins
are the least stable in a solution and easily precipitate. The isoelectric point of the
protein is highly dependent on the presence of salt ions in Lhc solution; al the same
time, its value is not affected by protein concentration.
With a shift in the acid-base balance of the body towards an increase in acidity
(decrease in pH), acidosis occurs. For example, with diabetes, there is an increase in
the production of ketone bodies (ketoacidosis). This is a dangerous condition for the
body, proteins lose their charge and their solubility decreases, while they can aggregate.

Protein solubility
Most proteins carry many charged groups on the surface; therefore, they arc
soluble in waLcr. Solubility is due to the presence of a charge, as well as the repulsion of
charged protein molecules. Besides,, the presence ofa hydration shell, i.c. environment
of a protein molecule with water dipoles and their interaction with polar and charged
groups on the surface ofa protein globule.

Protein isolation and purification methods

Obtaining individual proteins from biological material (tissues, organs, cell
cultures) requires sequential operations, including:
► crushing of biological material and destruction of cell membranes;
► fractionation of organelles containing certain proteins;
► protein extraction, (translating them into a dissolved state):
► separation of a mixture of proteins into individual proteins.
Protein separation methods arc based on the physicochemical properties of
proteins. The table (Table 1.3) shows the main methods of protein purification.

Ion exchange chromatography

The method is based on the separation of proteins that differ in total charge at certain
pH and ionic strength of the solution. When a protein solution is passed through a
chromatographic column filled with solid porous charged material, some ofthe proteins
are retained on it as a result of electrostatic interactions. As the stationary phase, ion
exchangers arc used — polymer organic substances containing charged functional
groups. There arc positively charged anion exchangers containing cationic groups and
negatively charged cation exchangers containing anionic groups. The choice of an ion
1.5. Physical Chemical Properties of Proteins 49

Table 13, The main methods of protein purification

1. Charge 1. Ion-exchange chromatography

2. Electrophoresis
3. Isoelectric focusing

2. Polarity 1. Adsorption chromatography

2. Paper chrom atography
3. Reverse-phase chromatography
4. Hydrophobic chromatography

3. Size 1. Dialysis and ultrafiltration

2. Gel electrophoresis
3. Gel tiltration chromatography
4. Uftracentrifigation

4. Specialty 1. Affinity chromatography

exchanger is determined by the charge of the protein released. So, to isolate a negatively
charged protein, an anion exchanger is used. Wien a protein solution is passed through
a column, the binding strength of the protein to the anion exchanger depends on the
number of negatively charged carboxyl groups in the molecule. Proteins adsorbed on
the anion exchanger can be washed oil (eluted} with buffer solutions with different salt
concentrations, most often NaCI, and different pH values. Chlorine ions bind lo the
positively charged functional groups of the anion exchanger and displace the carboxyl
groups of the proteins. At low salt concentrations, proteins weakly bound to the anion
exchanger elute. A gradual increase in salt concentration ora change in pH, which
changes the charge of a protein molecule, leads to the release of protein fractions, one
of which contains the desired protein (Fig. 1.31).

Low s= i efiution Hbgh-sall elution

buffer buffer

Fractions sequentially cotected

Fig. 131 The basic principles of ion exchange chromatography

50 Chapter 1. Tnb structure, properties and functions of proteins

Electrophoresis
The method is based on the property that, al a certain pH and ionic strength of a
solution, proteins move in an electric field at speed proportional to their total charge.
Proteins with a total negative charge move to the anode (+), and positively charged
proteins move to the cathode (-). Electrophoresis is carried out on various media:
paper, starch gel, polyacrylamide gel, etc. Unlike paper electrophoresis, where the
speed of proteins is proportional only to their total charge, in polyacrylamide gel the

r
speed of movement of proteins is proportional Lo their molecular weights (Fig, 1.32).

c
J2
n Well ■

E Langer
■C
u fragments
e
Cl

s-
£
u
fragments

1■
1
Fig. 132. The principle of separation of proteins using electrophoresis gel. Gel stained by Coomassie
brilliant blue reagent
1.5. Physical Chemical Properties of Proteins 51

The resolution of polyacrylamide gel electrophoresis is higher than on paper. To

delect protein fractions, strips of paper or poly acrylamide gel arc dyed. The colored
complex of proteins with a dye reveals the location of various fractions on the carrier.

Gel hitration chromatography

The method of protein separation using gel filtration chromatography is based on
the fact that substances with different molecular weights arc distributed differently
between the stationary' and mobile phases of the chromatographic column. Il is filled
with granules of a porous substance (Sephadex, agarose. etc.)T cross-links are formed
in the structure of the polysaccharide and granules writh. spores* are formed, through
which water and low molecular weight substances easily pass. Depending on the
conditions, granules with different pore sizes can. be formed. The stationary phase is
the liquid inside the granules into which low* molecular weight substances and proteins
with a small molecular weight can penetrate. A mixture of proteins deposited on. a
chromatographic column is washed (eluted) by passing a solvent through the column.
The largest molecules move along with the solvent front. Smaller molecules diffuse
inside the Sephadex granules and fall into the stationary phase for some time, as a
result of which their movement is delayed. The size of the pores determines the size of
the molecules that can penetrate into the granules (Fig. 1.33).

Fia 1.31 The Paste principles of qel filtration chromatography

Affinity chromatography
This is the most specific method for isolating individual proteins, based on the
selective interaction of proteins with ligands attached (immobilized) to a solid carrier.
As a ligand, a substrate or coenzyme can be used if any enzyme, antigens for the
isolation of antibodies, etc. arc isolated. A solution coma ini ng a protein mixture
is passed through a column filled with an immobilized ligand. Only a protein that
specifically interacts with it is attached to the ligand; all other proteins come out with
the eluate. The protein adsorbed on the column can be removed by washing it with
52 Cnaptef 1. Tn e structure, properties and functions of proteins

a solution with a changed pH value or a changed ionic strength. In some cases, a

detergent solution is used to break the hydrophobic bonds between the protein and
the ligand. Affinity chromatography is highly selective and helps to purify the secreted
protein thousands of limes (Fig. 1.34).

A
elution
A A A
» A .La
pH 2,0
A-L

Fig. 134. The basic principles of affinity chromatography

1.6. DISEASES ASSOCIATED WITH STRUCTURE AND FUNCTION

PROTEINS DISORDERS
The protein composition of an adult is more or less constant. However, some
changes in the content of certain proteins are possible depending on physiological
activity, food composition, etc. In diseases, the protein composition of tissues also
changes. These manifestations of the disease are called prolcinopathics.
In medicine, protcopathv refers to a class of diseases in which certain proteins
become structurally abnormal, and thereby disrupt the function of cells, tissues and
organs of the body. Often the proteins fail to fold into their normal configuration:
in this misfolded state, the proteins can become toxic in sonic way (a gain of toxic
function), or they can lose their normal function. The pnotcopathics (also known
as protein conformational disorders or protein misfolding diseases) include such
diseases as Alzheimer’s disease, Parkinson’s disease, amyloidosis, multiple system
atrophy and a wide range of other disorders. Separate primary and secondary
prole i. nopat hy.

Primary protein opathy

Hereditary p rotci nopat hy develops as a result of a damage in the genetic apparatus
of the individual. Any protein is not synthesized al all or is synthesized, but its primary
1.6. Diseases Associated witn Structure and Function Proteins Disorders 53

structure is changed. Examples of hereditary proicinopathy are hemoglobinopathies,

t hat have been discussed above. Depending on ihc role of the defective protein in the
vital activity of an organism, on the degree of disturbance of the conformation and
function of proteins, on the homo-or heterozygosity of an individual for this protein,
hereditary protcinopathies can cause diseases with varying degrees of severity, even
death before birth or in the first months after birth.

Secondary proteinopathy
Any disease is accompanied by a change in the protein composition of the body; i.c.
secondary proteinopathy develops. At the same Lime, the primary structure of proteins
is not disturbed, and usually there is a quantitative change in proteins, especially in
those organs and tissues in which the pathological process develops. For example,
pancreatitis reduces the production of enzymes necessary for the digestion of nutrients
in the gastrointestinal tract. I n some cases, acquired proteinopathy develops as a result
of changes in the conditions in which proteins function. So. when the pH of the
medium changes to the alkaline side < alkalosis of different natures, the conformation
of hemoglobin changes, its affinity for O2 increases, and the delivery of O? to tissues
(tissue hypoxia) decreases.
Sometimes as a result of the disease, the level of metabolites in the cells and blood
serum increases, which leads to the modification of certain proteins and the disruption
of their function. Thus, elevated blood glucose concentrations in diabetes mcllitus
lead to non-enzymatic attachment of glucose to proteins (glycosylation of proteins!.
An example is an increase in the level of glycated hemoglobin in red blood colls, which
increases its affinity for O, and reduces the transport of O, into tissues. Glycosylation
of proteins of the lens of the eye leads to its clouding and the development of cataracts.
In some cases, biochemical data on changes in the protein composition of the
blood or urine can be leading in the diagnosis. For example, in myeloma (malignant
degeneration of plasma cells that synthesize immunoglobulins}, Bcrts-Jones proteins
appear in the blood and urine, they’ arc present in low- concentrations in the blood of
healthy people. These proteins arc the light chains of immunoglobulin G. the synthesis
of which is enhanced in malignantly reborn ceils.

Conformational disorders
Some water-soluble proteins, when the conditions change, can acquire the
conformation of poorly soluble molecules capable of aggregation, forming fibrillar
deposits in cells called amyloid (from lai. amylum — starch). Like starch, amyloid
deposits arc delected by iodine staining of the tissue. This may occur
► wit h t h e overproduct ion of ccrtai n proici ns. as a resul t ofwrhich their concent rat ion
in ihc cell increases:
► when proteins enter the cells or form proteins in them that ean affect the
conformation of other protein molecules:
► upon activation of proteolysis of normal body proteins, with the formation of
insoluble fragments prone to aggregation:
► as a result of point mutations in the protein structure.
54 Cnaptef 1. Tn e structure, properties and functions of proteins

As a result of the deposition of amyloid in the organs and tissues, the structure
and function of cells arc disrupted, their degenerative changesand the proliferation of
connective tissue- cells arc observed. A disease called amyloidosis develops. Each type
of amyloidosis is characterized by a specific type of amyloid. Currently, more than
15 such diseases are described.
Alzheimer's disease is the most frequently observed p-amyloidosis of the nervous
system, usually affecting elderly people and characterized by progressive memory
disorder and complete personality degradation. In the brain tissue, 0-amyioid is
deposited — a protein that forms insoluble fibrils that disrupt the structure and
function of nerve cells. Protein p-amyloid is a product of a change in the conformation
of a normal protein of the human body, h is formed from a larger precursor by partial
proteolysis and is synthesized in many tissues. Protein p-amyloid, unlike its normal
precursor, which contains many a-heli cal regions, has a secondary P- fold structure
that can aggregate with the formation of insoluble fibrils, and is resistant to proteolytic
enzymes.
Parkinson’s disease was first described in 1817 by the English physician
James Parkinson (he called it «trembling paralysis*) and is one of the most
common neuro degenerative disorders. Most often, parkinsonism and other
ncu rod cgenc rad vc disorders (such as Alzheimer s disease) arc found in the elderly
and along with oncological diseases occupy a leading position among the causes of
death. Bui parkinsonism is not only a disease of the elderly: with the improvement
of diagnostic methods, there is more and more evidence that the disease affects
people under 40 years of age. As follows from the original name of the disease, its
characteristic symptoms arc motor disorders: trembling (tremors) of fingers, lower
jaw and tongue, head and eyelids, slowness and impoverishment of the pattern of
movements, stillness of the body, difficulty in starting and stopping movement,
impaired coordination, etc. Such disorders are caused by the flexibility of nerve
cells, primarily the loss of pigment-containing neuro n s of the which
produce the dopamine neuro transmit ter. Dopamine is the biochemical precursor
of norepinephrine and adrenaline. In many patients who died from Parkinson’s
disease, protein clusters are found during an autopsy in the ju&j/an/Kr (they
arc called Levy bodies by the name of the German pathologist who discovered them
in 1912). Parkinson’s disease develops as a result of disturbances in the functioning
of the chaperone and ubiquitin-protcasomc systems. Apparently, the situation is
as follows. Some damage in the neurons of the suistoitaj w/gra triggers a cascade
of reactions leading to the appearance of a large nu mber of improperly packaged
proteins.
1.6. Diseases Associated with Structure and Function Proteins Disorders S5

Review tests
1. Choose one correct answer. I IBS in contrast to I IbA:
A. Has a 6-posilion a-chain Vai.
B. Contains 2a and 2.y chains.
C. Deoxyform is poorly soluble in water.
D. Has a high affinity for (\.
E. In the active center there is an amino acid substitution.
2. Choose one correct answer. Isoelectric point of proteins:
A. The amount o! protein charge.
B. The ratio of polar and nonpolar amino acid radicals.
C. The pH value at which the protein has a charge equal to zero.
D. The pH value at which the protein is best soluble in water.
E. The charge of the protein at which it is most actively moving in the electric field.
3. Match the figure with the letter. Hemoglobin molecule:
A. a chain Hb.
B. p chain Hb.
C. .Active center.
IX Central cavity.
E. Domain.
■4. Match the figure with the letter. Protein
separation methods:
1. Ci cl filtration.
2. Electrophoresis.
3. .Affinity chromatography.
The principles of separation by difference:
A. Charges.
B. Solubility in water.
C. Molecular weight.
D. Sedimentation rate in solution.
E. .Affinity for a specific ligand.
5. Choose all correct answers. The affinity of I I B to oxygen decreases with:
A. An increase in rhe concentration of protons.
B. A decrease in the concentration of protons.
C. .A decrease in the concentration of 2,3-BPH.
D. An increase in the concentration of 2.3-B PH.
E. Sequential cleavage of oxygen molecules.
6. Match the figure with the letter. Functional parts of the oligomeric protein:

Active site

1
56 Cnaptef 1. Tn e structure, properties and functions of proteins

A. Subunit.
B. An allosteric center.
C. Active center.
D. An oligomer.
E. An effector.

Situational problems
1. In high altitude conditions, climbers usually led the clinical signs of hypoxia:
headache, shortness of breath, nausea, increased heart rate. However, after
2 days of rest in the base camp, the symptoms disappear. How docs the body
adapt to high altitude conditions and increase oxygen delivery to tissues?
2. The fetal HbF has a higher affinity for oxygen than the mother HbA. What
structural features of these proteins determine their difference in the ability to
bind oxygen and what role docs 2,3-bisphosphoglyccrate play in this? What is the
physiological meaning of t he different affinities of these Hb forms for oxygen?
3. The patient went to the clinic with complaints of dizziness, shortness of breath,
palpitations and pain in the limbs, which sharply worsened after a short rest in
the mountains. A reduced number of red blood cells were found in the patient's
blood, as well as immature crescent-shaped cells and red blood cells. What arc
the causes of this pathology? Why did the disease worsen in conditions of low-
partial pressure of oxygen?
 Chapter 2
ENZYMES

2.1 Enzymes as Catalysts. Active Site of the Enzy me

22. Vitamins. Cofactors and Coenzymes
2.3. Enzymes Classification and Nomenclature
2.4. Regulation of Enzymes and Metabolic Pathways. Inhibition of Enzymes.
Drugs as Inhibitors of Enzymes
2.5. Clinical Applications of Enzymes. Plasma Proteins and Enzymes in the Diagnosis
of Diseases

2 J. ENZYMES AS CATALYSTS. ACTIVE SITE OF THE ENZYME

The transfer mat ions of various molecules in the cells of the body catalyze enzymes.
Enzymes are proteins thaL have a specific primary, secondary, tertiary; and sometimes
quaternary structure.
Enzymes are a group of proteins that have an ability to accelerate chemical
reactions. Enzymes distinguish three unique properties from other catalysts:
► high efficiency of action;
► action specificity;
► ability to be regulated by other molecules:
By their function, enzymes arc biological catalysts. The essence of the action of
enzymes, as well as inorganic catalysts, is;
► in the activation of reactive molecules:
► in dividing the reaction into several stages, the energy barrier of each of which is
lower than that of the overall reaction.
However, enzymes will not catalyze energy-impossible reactions: they only
accelerate those reactions that can take place under these conditions. It is known
that for the implementation of a chemical reaction, it is necessary that the reacti ng
substances have total energy higher than the value called the energy barrier of the
reaction. Enzymes arc proteins that bind to a molecule, or substrate, to modify it
and lower the energy required to make it react. The raLc of reaction is given by the
Arrhenius equation. In this figure, the plot of energy versus the progress of a reaction
is shown (Fig. 2.1). Reactants have higher energy than products. The energy of the
reactants increasesand then decreases to the final product energy. The highest point
in the curve represents the energy of the intermediate state in the reaction. The energy
required to achieve the intermediate state is the activation energy of the reaction.
Enzymes fewer the activation energy of a given reaction, shown by the blue curve.
53 Chapter 2. Enzymes

Figu 2.1 Graphs ol me reaction energy in me absence anti presence of the enzyme

The similarities and differences of enzymes and inorganic

catalysts

Similarity
1. Catalyze only energetically possible reactions;
2. Do not change the direction of the reaction:
3. Accelerate the onset of reaction equilibrium, but do not shift it;
4. Not consumed during the reaction.

Differences
1. The velocity of the enzymatic reaction is much higher:
2. High, specificity;
3. Mild working conditions (intracellular);
4. The ability to control the reaction rate;
5. The velocity of the enzymatic reaction is proportional to the amount of enzyme.
The catalytic power of enzymes (the ratio of the reaction rate in the presence of
a catalyst to the reaction rate without a catalyst) is in the range from 106 to 1014, so
they can provide rapid processes, such as heartbeats or conduction of nerve impulses.

Enzymatic catalysis
Enzymes reduce the activation energy through a process called catalysis. A
biochemical reaction when an enzyme is present is called a catalyzed reaction.
Catalysis can happen in di fie rent ways. Enzymes can use the transfer of protons or
electrons to the reactants to modify the state of the reactants. Enzymes also use electric
charges to stabilize the state of the reactants. Enzymes, however, do not modify the
final products of the reaction.
2.1. Enzymes as Catalysts. Active Site cf the Enzyme 59

The following steps arc distinguished in the enzymatic reaction (Fig. 2.2-23):
12 3 4
E + S -<------ ► E-S <------ ► E-X ■*------ ► E-P * * E +P

The enzyme changes shape.

The 5ttJSlra?B5 tmd to be The product is released When
Oenzyme's active site. Q which catalyzes the chemical
reaction between be substrates.
Gthe reaetton is comptele.

Fig. 22.2.3. The steps o f enzymatic reaction

Steps of enzymatic catalysis:

1) the attachment of the substrate (S) to the active site of the enzyme (E) with the
formation of the enzyme-substrate complex (E-S):
2) transformation of an enzyme-substrate complex into one or several transitional
complexes (E-X) in one or several stages;
3) transformation oft he transitional complex into the enzyme-product complex (E-P):
4} the separation of the final products from the enzyme.

Structural and functional organization of enzymes

All enzymes arc proteins and have all the properties of proteins. Therefore, like
proteins, enzymes arc divided into simple and complex.
Simple enzymes consist only of amino acids — for example, pepsin, trypsin,
lysozyme (Fig. 2.4}.

Fig. 2.4. Structure ot the simple enzyme

60 Chapter 2. Enzymes

Complex enzymes (holoenzymes) arc composed of a protein pan consisting of

amino acids — an apoenzyme and a non-prolein pari — a cofactor (Fig. 2.5).

Apoenzyme Cofactor Holoenzyme

(protein portion), (nonprotein portion), (whole enzyme).
inactive activator active

Fig. 2.5. Structure of the complex enzyme

The cofactor, in turn, can be called a coenzyme (NAD-F, NADP+, FMN, FAD,
biotin) ora prosthetic group (heme, oligosaccharides, ions of metals Fc2\ Mg1*, Ca2',
Zn2'). If Lire connection between the cofactor and the protein is strong, then, in this
case, it is said that there is a prosthetic group, but if a vitamin derivative is used as a
cofactor, it is called a coenzyme, regardless of bond strength. For the implementation
of catalysis, a full-fledged complex of the apoprotein and a cofactor is necessary;
separately, they cannot carry out catalysis. A cofactor is pari of the active center, is
involved in the binding of the substrate or in its transformation. Examples of complex
enzymes are succinate dehydrogenase (contains FAD), aminotransferases (contain
pyridoxal phosphate), peroxidase (contains heme), lactate dehydrogenase (contains
Zn2*), amylase (contains Ca2). Like many proteins, enzymes can be monomers, i.c..
consist of one subunit and polymers consisting of several subunits.
As part of the cnzvmc active site, isolated areas that perform a different function.
(Fig. 2.6).
Active site (active center) — a combination of amino acid residues (usually 12—16)
providing direct binding lo the substrate molecule and performing catalysis. Amino
acid radicals in the active center can be in any combination, with the amino acids
located far from each other in a linear chain located nearby. In the active center, there
arc two sites — binding site and catalytic site. Binding site (contact, anchor) — is
responsible for binding and orientation of the substrate in the active center. Catalytic
site — is directly responsible for the implementation of the reaction.
Enzymes containing several monomers may have several active centers in the
number of subunits. Also, two or more subunitscan form one active center. I n complex
enzymes, functional groups of the cofactor are necessarily located in the active center
(Fig. 2.7).
2.1. Enzymes as Catalysts. Active Site of the Enzyme 61

Protein structure
Stafford to support and
position active site

Active site
Binding sites Catalytic site

Bind and orient Reduce chemical

suDstrate(s) activation energy

Fig. 2.6. Structure of the active site of the enzyme

WithoLin the cofactor

Cofactor binding
attached, the protein
activates the protein.
is not active.

Fig. 2 J. The structure of the active site of We complex enzyme. A coenzyme or cotactor is involved
in its formation

Allosteric center (alios —alien) is the center of the activity of the enzyme regulation,
which is spatially separated from the active center and is not available for all enzymes.
Ilinding to the allosteric center of any molecule (called an activator or inhibitor, as
well as an. effector, modulator, regulator) causes a change in the protein-enzyme
configuration and, as a consequence, the rate of the enzymatic reaction {Fig. 2.8).
62 Chapter 2. Enzymes

(a) Allosteric inhibition

Distorted ■ Substrate

(b) Allosteric activation

Rg. 2.8. Allosteric site of the enzyme allows to regulate the activity of the enzyme

Allosteric enzymes arc oligomeric proteins, active and regulatory centers are
located in diflerent subunits. Such a regulator may be the product of this or one of the
subsequent reactions, the substrate of the reaction, orothersubstan.ee.

Enzyme specificity
One of the important characteristics of enzymes is their high specificity, it lies
in the fact that each, enzyme catalyzes the transformation of a particular substrate
or a group of substrates that arc similar in structure. The specificity of the action of
enzymes determines the directional metabolism in the body.
Specificity, i.c., the high selectivity of the action of enzymes is based on the
complementarity, it’s mean structural and chemical correspondence of the substrate
structure and the active center of the enzyme (Fig. 2.9).

Fig 2.9. Schematic representation of the specificity ol Uie enzyme

2.1. Enzymes as Catalysts. Active Site cf tne Enzyme 63

Absolute specificity — the enzyme produces catalysis of only one substrate. For
example, urease cleaves only urea and docs not act on other compounds. Il acts on
urea, but not at thiourea, mcthylurca. biuret.
Group or relative specificity - catalysis of substrates with common structural
features, i.c., in the presence of a specific bond or a chemical group. For example,
pancreatic lipase hydrolyzing triacylglyccrols (TAG) with different fatty acid
composition to 2-monoacylglycerols (2-MAG) and fatty acids breaks down ester
bonds in various food fats.

O
Urease
h2n c — nh2 + h2o CO2 + 2NH3
urea

o S O O
II
II I 11
HSN- C- NHCHj h2n -c- ■ nh2 h2n — C —NH— C — NFfe
Methylurea Thiourea Biuret

Pepsin catalyzes the breakdown of a peptide bond formed by amino groups of

aromatic amino acids.
O
II
H2C—O—C—R H2C —

h2o --O
HC—O— R‘
Lipase upase
O
|l
H2C—-O—C—R" H2C — O— H2C—OH
Triacygiyceroi Diacygfyceroi Monoacylglycerol

Pepsin

Polypeptide Polypeptide fragments

R and R1 = Leu. Phe, Trp, and Tyr (preferred); also hydrolyzes esters
64 Chapter 2. Enzymes

Stereo specificity — catalysis of only one of the stereoisomers of a molecule. For

example, specificity for L- or D-amino acids — almost all human enzymes interact
with L-a mi no acids, specificity for cis- and trans-isomers. Aspartase reacts only with,
the trans-isomcr— fumarate, but not with the maleate (cis-isomer).

H NH./ H
I Aspartase
C— C---- CO< C + NH/
I I
H H CO2“

L Aspartate Fumarate
Catalytic specificity or specificity of transformations — the specificity of the
transformation paths lies in the fact that a single substrate under the action of
di fie rent enzymes can turn into products that differ in the structure and their role
in metabolism. For example, the transformation of malate by two different enzymes:
malate dehydrogenase and malic enzyme.

Two models of the enzymes specificity. There arc two main models explaining the
specificity of enzymes. In 1890 the chemist Emil Fischer proposed that the substrate
of an enzyme fits into the enzyme’s active site, the physical location on an enzyme
where the reaction takes place, to form an enzyme-substrate complex. The analogy he
used was of a lock and key. The key (substrate) has a specific shape (arrangement of
functional groups and other atoms) that allows it and no other key to fit into the lock
(the enzyme). A lock-and-kcy model explains absolute specificity (Fig. 2.10).

Lock-ana-key model Enzyme-substrate

complex

R& 2.10. Ltrch-and-key model specificity of me enzymes

2.1. Enzymes as Catalysts. Active Site of tne Enzyme 65

In 1958, Daniel E. Koshland Jr. modified the loek-and-key model by proposing

that binding of the substrate to the enzyme alters the configuration of both, providing
a better fit. The induced-fit model explains group specificity (Fig. 2.1 I L

induced fit model Enzyme-substrate

complex

Fig_ 2.11. The i ndueed-fit mode I specificity of the enzymes

Enzyme kinetics
Under optimal conditions, the enzyme activity depends on:
* the amount of enzyme (E);
> substrate amounts (S):
> the amount of product (P);
* cofactor concentrations:
> presence of activators or inhibitors.
Temperature and pH also affect the rate of enzymatic reactions.

Dependence on the enzyme amount

With an increase in the number of enzyme molecules, the reaction rate increases
continuously and is directly proportional to the amount of the enzyme, since more
enzyme molecules produce more product molecules (Tig. 2.12).

Fig. 2.12. The dependence of the enzymatic reaction rate on the concentration of me enzyme
66 Chapter 2. Enzymes

Dependence of the reaction rate on the substrate concentration

With increasing substrate concentration, the reaction rate first increases, because new
and now enzyme molecules are connected to the catalysis ofthe added substrate molecules.
Those, the rale of accumulation of the product increases, and this means an increase in
the enzyme activity of the. Then there is a saturation effect (plateau on the curve), when
all the molecules of the enzyme arc occupied by substrate molecules and continuously
catalyze, here the reaction rate is maximum. In some cases, with a furl her increase in the
concentration of the substrate between its molecules, there is a competition for the active
center of the enzyme and the enzyme activity (reaction rate) decreases (Fig. 2.13).

o
Substrate concentration's

Fig. 2J3. The dependence of the enzymatic reaction rate on die concentration of me substrate

The general theory of enzymatic kinetics and the dependence of enzyme activity
on the substrate described by L. Michaelis and M.L. Menton, gives it in their equation.
The Michaelis-Men ten equation shows the relationship between the maximum possible
speed, the real reaction rate, the Michaelis constant, and the substrate concentration.
Km (the Michaelis constant) is visible in. the graph as the concentration of a substrate
al which the initial velocity ishalfoft.be maximum velocity (Fig. 2.14).

Fig: 2.14 Delernri nation of Km and Vjiai according to the graph of dependence of tne enzymatic
reaction rate on the concentration of the substrate
2.1. Enzymes as Catalysts. Active Site cf the Enzyme &7

In the Michaelis-Men ten equation, and Km play an important role.

Michael is-Men ten equation:
^max I c'l
V0= -----------
Km-HS]

where: Vfl = the velocity al any time: [S]l = the substrate concent ration at this
limejV^ = the highest under this set of experimental conditions (pH, temperature,
etc.); Km = the Michaelis constant for the particular enzyme being investigated.
Maximal Velocity (V^j: Increasing the substrate concentration indefinitely docs
not increase the rate of an enzymc-cataiyzed reaction beyond a certain point. This
point is reached when there arc enough substrate molecules to completely fill (saturate)
the enzyme’s active sites. The maximal velocity, or V^, is the rate of the reaction
under these: conditions. Vnia* reflects how fast the enzyme can catalyze the reaction.
Michaelis Constant (Km): Enzymes have varying abilities to bind their substrates
(affinities). An enzyme’s Km describes the substrate concentration al which half the
enzyme’s active sites arc occupied by substrate. A high Km means that a lot orsubsiratc
must be present to saturate the enzyme, meaning the enzyme has a low affinity for the
substrate. On the other hand, a low Km means that only a small amount of substrate is
needed to saturate the enzyme, indicating a high affinity for substrate.
These constants arc important to understand enzyme activity in general, as well as
to understand the effects of different types of enzyme inhibitors.

The dependence of the reaction rate on temperature

The dependence of enzyme activity (reaction rate) on temperature that looks like
a bell-shaped curve with maximum velocity at the optimum temperature (Fig. 2.15).
The law on increasing the reaction rate by 2-4 times with an increase in tempera Lure
of ID °C is also valid for enzymatic reactions, but only up to 55-60 °C, i.c.r up to
protein denaturation tempera Lures.

Fig. 2.15. Graph of reaction rate on the temperature

68 Chapter 2. Enzymes

Along with this, as an exception, there arc enzymes of some microorganisms

that exist in the water of hot springs and geysers. At lower temperatures, the activity
of enzymes decreases, but does not disappear altogether. An i[lustration may be
hibernation of some animals (gophers, hedgehogs), who’s body temperature drops to
3-5 ”C. This property of enzymes is also used in surgical practice when performing
operations on the chest cavity when the patient is subjected to cooling to 22 C.

Dependence of reaction rate on pH

The dependence is also described by a bell-shaped curve with a maximum speed at
the optimum pH value for this enzyme (Fig. 2.16).

Fig. 2.16 Graph of reaction rate on Ute pH

This feature of enzymes is essential for the organism in its adaptation to changing
external and internal conditions. Shifts in pH outside and inside the cell plays a role in
the pathogenesis of diseases, changing the activity of enzymes of different metabolic
pathways. For each enzyme, there is a certain narrow range of pH. which is optimal for
the manifestation of its highcractivity. For example, the optimum pH values for pepsin
are 1.5-2.5, trypsin 8.0-8.5, amylase of saliva 7.2, arginase 9.7, acid phosphatase 4.5-
5.0, succinate dehydrogenase 9.0 (Fig. 2.I7, Table 2.1).

Kg. 2.17. Optimum pH values for various enzymes

2.1. Enzymes as Catalysts. Active Site of tne Enzyme 69

Table 2.1. Optimum pH values tor various enzymes with different location and substrates

Enzyme Location Substrate Optimum pH

Pepsin Stomach Peptide bonds 2

Urease Liver Urea 5

Sucrase Small intestine Sucrase 6_2

Pancreas: mylase Pancreas Amylase 7

Trypsin Small intestine Peptide bonds 6

Arginase Liver Arginine 9.7

Units of measurement of enzyme activity

A characteristic of the activity of enzymes is the rate at which they catalyze a
particular reaction. It is measured by the rale of transformation of the substrate or the
rate of accumulation of the reaction products, it is necessary to measure the initial rate
of transformation, and not the amount of substrate convened over a certain period: of
time.

Enzyme activity
The Commission on Enzymes of the International Biochemical Union gives an
idea of the standard unit of activity. The unit of activity (U) is the amount of enzyme
that catalyzes the conversion of one micromole of substrate per minute under standard
conditions (at optimum pH, with an excess of substrate, at a temperature of 37 or
20 °C).
Enzyme activity = moles of substrate convened per unit time = rate x reaction
volume. Enzyme activity is a measure of the quantity of active enzyme present and
is thus dependent on conditions, which should be specified. The SI unit is the kata!,
I katal = I mol s but this is an excessively large unit. A more practical and commonly
used value is enzyme unit (U) = 1 pmol min ]. I U corresponds to 16.67 nanokatals.
An increased amount of substrate will increase the rale of reaction with enzymes;
however, once past a certain point, the rate of reaction will level out because the
amount of active sites available has stayed constant.

Specific activity
The specific activity ol’an enzyme is another common unit. This is the activity of an
enzyme per milligram of total protein (expressed in pmoi min 'mg '). Specific activity
gives a measurement of enzyme purity in the mixture. It is the micromoles of product
formed by an enzyme in a given amount of time (minutes) under given conditions per
milligram of total proteins. Specific activity is equal to the rate of reaction multiplied
by the volume of reaction divided by the mass of total protein. The SI unit is katal
kg but a more practical unit is pmol mg 1 min '. Specific activity is a measure of
enzyme process! vity, at a specific (usually saturating) substrate concentration, and is
usually constant for a pure enzyme. The specific activity should then be expressed as
70 Chapter 2. Enzymes

limol min 1 mg 1 of active enzyme. If the molecular weight of the enzyme is known,
the turnover number, or pmol product per second per pmol of active enzyme, can be
calculated from the specific activity. The turnover number can. be visualized as the
number of times each enzyme molecule carries out its catalytic cycle per second.

Classical units:
Unit of enzyme activity:
pmol substrate iransformed/'min = unit

Specific activity:
pmol subst rate/min-mg E = unil/mg E

Review tests
1. Match the figure with the letter;
On the image, which letter represents the enzyme a), the
substrate b), the product of the reaction c)?
A.
B
C
2. The part of the enzyme where the substrate binds is called
the;
A. Active site.
B. Catalyst.
C. Inhibitor.
D. Large subunit.
3. Enzymes have a distinct advantage over non-biological catalysts because they:
A. Arc made of protein.
B. Arc very efficient, specific, and sensitive to control
C. Arc all allosteric.
D. Can't be denatured by heating.
4. Choose all the correct answers. In the process of enzymal k catalysis occurs: E + S >
ES > EX > EP > E + P:
A. Establishing rhe induced correspondence between the substrate and the active
center of the enzyme.
B. The formation of covalent bonds between the substrate and the active center.
C. A change in the conformation of the enzyme.
D. The formation of an enzyme-subst rate complex.
E. Destabilization of bonds in the substrate molecule.
5. Find a match. Specificity:
1. Absolute.
2. Relative.
3. Catalytic.
Enzyme:
A. Catalyzes the transformation of the substrate in one of the ways of transformation.
B. Interacts with only one substrate.
C. Catalyzes several different transformations of the same substrate.
2.1. Enzymes as Catalysts. Active Site of tne Enzyme 71

D. Can interact with a group of si mi I ar substrates.

E. Interacts with only one of the stereoisomers fora given substance.
6. Choose all the correct answers. The Km (Michaelis constant) of an enzyme-catalyzed
reaction:
A. Is a measure of the turnover rate of the enzyme.
B. k a measure of the affinity of the enzyme for its substrate.
C. Has a higher numerical value when the enzyme has high affinity for its substrate.
D. Has a lower numerical value when lhe enzyme has high affinity for its substrate.
E. Is Lhe substrate concentration when the reaction rate is half of the maximum rate.
7. Analyze reaction energy profile.
4 L

Reaction

Parameters, profile analysis:

A. The total energy of the system.
B. Activation energy.
C. ES complex.
D. The energy of the products.
E. Reaction without enzyme.

Situational Problems
1. The optimal conditions for salivary lysozyme (hydrolyzing glycoproteins of bacterial
wail) are 37 C — temperature and pH is 5.2. Explain the decrease in this enzyme
activity if the temperature will rise up to 60 T and pH will be changed to 8.0. To
answer the question:
a) draw the graph of the velocity dependency on temperature and pH:
b) calculate the relative enzyme activity if 10 mg of lysozyme catalyzes the
formation of 5 pM of the product per 2 minutes.
2. Consider lhe enzy matic reaction scheme: Asparagine + H20 > Aspartate + NH3:
a) calculate the specific activity of the enzyme, if in 30 seconds as a result of
a reaction involving 3 mg of the enzyme under optimal conditions (pH 8.0,
37 *C) 75 pmol aspartate is obtained;
b) describe the masons lor the decrease: in enzyme activity after incubation for
10 minutes at 70 °C (provide an appropriate graph).
72 Chapter 2. Enzymes

3. The figure below shows the reaction velocity catalyzed by an enzyme as a function
of the enzyme substrate concentration. Such a relationship is typically referred
to as a dose-response relationship. Based on the data shown, what is the Km
(Michaelis constant) of this enzyme for its substrate? Note that the reaction
velocity is normalized to the maximum velocity (VmHj. Therefore, in the plot
shown, VmaK = 100%:

a) 2.5 nM;
b) 5.0 nM:
c) 10.0 nM:
d) 20.0 nM.

22. VITAMINS. COFACTORS AND COENZYMES

Vitamins arc a group of low-molecular weight substances of various nature,
which arc not produced by the organism and arc necessary for biochemical
reactions that ensure growth, survival, and reproduction of the organism. Vitamins
usually participate in the role of coenzymes and prosthetic groups in the work of
enzymes.
Everyone knows that vitamins arc necessary for our body. We get vitamins
from food, vegetables, and fruits. Often, we take synthetic vitamins, unfortunately,
synthetic vitamins arc much less effective for the body. They do not easily cross from
the intestines into the blood stream, but arc instead eliminated from the body. And
if they are able to be absorbed, synthetic vitamins and supplements may not be used
efficiently by the body; because of variations in their chemical structure from whole
food vitamins.
2.2. Vitamins. Cofactors ano Coenzymes 73

Vitamins classification
Vitamins were originally classified according io their solubility in water or fats, and
later as more were discovered, they wore also classified alphabetically. The fat-soluble
vitamins arc A, D, E,and K: the B complex and C vitamins arc water soluble. A group
of substances that decrease blood capillary fragility, called the vitamin P group, are no
longer considered to be vitamins.
1. Pat-soluble vitamins: D (calciferol), E (tocopherol), F (polyunsaturated fatty
acids), K (naphthoquinone), A (retinol). The function of fat-soluble vitamins
can be coenzyme (vitamin K), antioxidant ( vitamins A and E), or hormonal
(vitamins A and D).
2. Water soluble vitamins: B, (thiamine), B3 (riboBavin), B3 (nicotinamide),
B. (pantothenic acid), B (pyridoxine), B, (BC, folic acid), B|2 (cobalamins),
H (B7, biotin), C (vitamin C).
3. Vitamin-like substances:
* fat soluble — Q (ubiquinone):
* water soluble — B4 (choline), P (bioflavonoids), Bg. (inositol), B|o (para-
anti no benzoic acid), Bn (BT, carnitine).

General properties of vitamins

Vitamins arc not formed in the body: their biosynthesis is carried out outside the
human body, i.c., vitamins must come from food. An exception is vitamin PP, which
can be synthesized from tryptophan, and vitamin D (choiccalciferol), synthesized
from cholesterol.
Vitamins do not serve as an energy source. The exception is vitamin F.
Vitamins arc essential for all life processes and arc biologically active in small
quantities.
When they enter the body, they affect biochemical processes occurring in any
tissues and organs, i.e., they arc not organ specific.
In high doses, vitamins can be used for medical purposes as non-specific agents.
For example, with diabetes mellitus — B B... Bb, with colds and infectious diseases —
vitamin C, with bronchial asthma — vitamin PP, with ulcers of the digestive tract —
vitamin-like substance U and nicotinic acid, with hypercholesterolemia — nicotinic
acid.
The table below shows some vitamins and their functions, the names of the
coenzymes they form, and the reactions in which they participate (Table 2.2).
74 Chapter 2. Enzymes

Tatle 22. Characteristics of vitamins, their coenzymes and functions

B1 (ttiiamin) Thiamine-pyrophosphate (TPP) decarboxylation

B.? (riboflavin) Flavin-aden ine-d inucle otide (FAD) Hydrogen transfer

Eh (nicotinic acid, niacin) Nicotinamide-adenine dinucleobde iNAD+) Hydrogen transfer

B4 (folic acid] Tetrahydrofolic acid Formylation

B, (pantothenic acid} Coenzyme-A Transfer an acyl group

B- (pyridoxine) Piridoxal-phosphate Transamination

B1? (cobalamin) Cobamide (Btf) Transfer ol carboxyl group

H (biotin) Biotin Carboxylation

C (ascorbic acid) none Oxidation-reduction

Formulas and properties of the most important vitamins

Below we look, at more detail al sonic of the most important vitamins.

Vitamin Bi (Thiamine)
Vitamin B, or thiamine, enables the body to use carbohydrates as energy. It is pan
of thiamine di phosphate (TOP), which is the coenzyme of transketolase, the enzyme
of [he pentose phosphate pathway. Thiamin serves as a cofactor fora series of enzymes
in different metabolic pathways and is required for the production of ATP (Ch. 5),
ribose, NAD, and DNA. Il is essential for glucose metabolism, and it plays a key role
in nerve, muscle, and heart function. Vitamin B, is a water-soluble vitamin, as arc all
vitamins of the B complex.
2.2. Vitamins. Cofactors ano Coenzymes 75

nh2

A deficiency of vitamin l?( commonly leads to beriberi disease, a condition that

features problems with the peripheral nerves and wasting. Weight loss and anorexia
can develop. There may be mental problems, including confusion and short-term
memory loss. Muscles may become weak, and cardiovascular symptoms can occur,
for example, an enlarged heart ( hypertrophy).

Vitamin (Riboflavin)
Vitamin Bp or riboflavin, is one of eight I? vitamins that are essential for human
health. It can be found in grains, plants, and dairy products. Riboflavin (Vitamin Bj
is the precursor of two coenzymes known as flavin mononucleotide (FMN) and
flavin adenine di nucleotide (FAD). Both arc essential for tissue respiration and the
generation of energy front the metabolism of carbohydrates, amino acids and fats.
Riboflavin is vital for normal reproduction., growth, repair and development of
body tissues, including the skin, hair, nails, connective tissue and immune system.
Riboflavin is mainly convened into FMN and FAD in the small intestine, liver, heart,
and kidneys.

FSboflavin Flawn mononucleotide (FMN’! Flavin adenine dhudeolide {FAD J

76 Chapter 2. Enzymes

FAD + 2H*>2e"-FADH2

Riboflavin deficiency (also called ariboflavinosis) results in stomatitis, chapped

and fissured lips (cheilosis), and in llam mat ion of the comers of the mouth (angular
stomatitis). Due to interference with iron absorption, even mild to moderate riboflavin
deficiency results in anemia with normal cell size and normal hemoglobin content
(i.c., normochromic normocytic anemia). Deficiency of riboflavin during pregnancy
can result in. birth defects including congenital heart defects and limb deformities.
Prolonged riboflavin insufficiency is also known to cause degeneration of the liver and
nervous system.

Vitamin B3 (Niacin)
Vitamin Br also known as niacin, plays a key role in skin, digestive, and mental
health, and supports the functions of more than 200 enzymes in the body. Vitamin B3
is a combination of two chemicals: nicotinic acid and nicotinamide. The body breaks
these chemicals down to produce two additional chemicals: NAD and NADP.
NAD and NADP arc coenzymes of most dehydrogenases, participate in the
reactions:
► synthesis and oxidation of carboxylic acids:
► cholesterol synthesis:
► exchange of glutamic acid and other amino acids;
► carbohydrate metabolism: pentose phosphate pathway, glycolysis;
► oxidative decarboxylation of pyruvic acid;
► Krebs cycle, etc.
NAD and NADP play a role in a variety of chemical reactions inside the body
and also support cell metabolism. So, people who don’t get enough vitamin B3 can
experience a range of health problems and symptoms, ranging from minor to I ire-
threatening. Vitamin B3 deficiency can disrupt dozens of processes in the body and can
lead to a disease called pellagra.
2.2. Vitamins. Cofactors ano Coenzymes 77

Vitamin Bs (Pantothenic acid)

Vitamin B. is also known as pantothenic acid, or Pantothenate. The word
pantothenic comes from the Greek ^pantou,* meaning everywhere. Nearly all foods
contain small quantities of pantothenic acid. Vitamin B5 has a role in. synthesizing
coenzyme A. Coenzyme A is involved in the synthesis of fatty acids and is important
for converting foods into fatty acids and cholesterol. Coenzyme A is also needed for
the creation of sphingosine, a fat-like molecule that helps deliver chemical messages
inside the body's cells. The liver needs Coenzyme A to metabolize some drugs and
toxins safely.

■
■
i
■
cooh!

ch3 h

Pantoic acid part. p-Alanine pari

J
Pantothenic acid

■ i
I

Coenzyme A
78 Chapter 2. Enzymes

Vi Lamin B deficiency is extremely rare in people as pantothenic acid is found

in nearly all foods. A healthy and varied diet should provide a person with enough
vitamin B.. Clinical trials have shown, however, that a deficiency may lead to tiredness,
apathy, depression, vomiting, muscle cramps, hypoglycemia, upper respiratory
infections. A deficiency of B5 can cause the increased sensitivity lo insulin.

Vitamin H or B? (Biotin)
Biotin is a water-soluble vitamin, also called vitamin B? and formerly known as
vitamin II or coenzyme R. It is involved in a wide range of metabolic processes,
both in humans and in other organisms, primarily related to the utilization of fats,
carbohydrates, and amino acids. Biotin is important in fatty acid synthesis, branched-
chain amino acid catabolism, and gluconeogenesis. Biotin is involved in the transfer
of OO2 cither from HCO3 (carboxylation reaction) or from R-COOH (trans­
carboxylation reaction). Such a reaction is necessary for the synthesis ofoxaloacctale —
biotin is a part of pyruvate carboxylase complex, which ensures the maintenance of the
activity of the tricarboxylic acid (TCA) cycle and gluconeogenesis. In the synthesis of
fatty acids — biotin is in the composition of acetyl-CoA — carboxylase a key enzyme
of th is process.

(CH2)4—COOH

Hypovitaminosis of vitamin H in humans almost never occurs. The experiment

reveals dermatitis, fat secretion of the sebaceous glands of the skin (seborrhea), nail
damage, hair loss, anemia, anorexia, depression, fatigue, drowsiness.

23. ENZYMES CLASSIFICATION AND NOMENCLATURE

Enzymes have been known to people for a very long time. Therefore, many of
them have trivial and common names. A trivial name is a name that has developed
historically, for example, pepsin, trypsin, papain, bromelain, and chymosin, don't
give information about the substrate, product or chemistry of reaction. The common
names for some enzymes (more often for hydrolases), the termination of *-ase* —
invertase, urease, amylase, lactase, lipase — is added to the name of the substrate.
Common names don’t describe the chemistry of the reaction. Nevertheless, such
enzymes have a systematic name.
In 1961 in Moscow, the 5lh International Biochemical Union adopted a modern,
classification of enzymes. In accordance with this classification, all enzymes are
divided:
2.3. Enzymes Classification and Nomenclature 79

» into classes — by type of catalyzed reaction;

► each class is divided into subclasses — by die nature of the chemical group under
attack;
► subclasses arc divided into sub subclasses — by the nature of the attacked
connection or by the nature of the acceptor.
There arc 6 classes of enzymes:
» class 1 — Oxidoreductases;
► class 2 — Transferases;
► class 3 — Hydrolases:
► class 4 — Liases;
» class 5 — Isomerases;
► class 6 — Ligases.
Each enzyme is assigned a four-digit classification, number, including a class, a
subclass, a sub-subclass, and a serial number in the subclass.
For example, alcohol dehydrogenase has EC (Enzyme Commission)
number 1.1.1.1. — it is an oxidorcductasc, acts on. the donor OH group with NAD as
an acceptor with the first serial number in its subclass: lactate dehydrogenase - EC
1.1.1.27, acts on the donor OH group with NAD as an acceptor with the sequence
number 27 in its sub-subclass.
The table below (Table 2.3) shows the classes of enzymes, the main representatives
and types of catalyzed reactions.
Table 2 J. Enzyme classes and types of chemical reactions

1. Oxidoreductases Oxidases AJ+-B,?------ >AJ4-B<s Alcohol + NAD

(Transfer ot electrons) Reductases X Alcohol dehydrogenase
Dehydrogenase Aldehyde + NADH?

2. Transferases Transaminase A-X4-B -> A+B-X Glucose + ATR

(Transfer o! functional Transketolase X Glue, glycokinase or
groups) Transaidolase hexokinase
Glucose-6-Phosphate + ADP

3. Hydrolases Amylases A—B-i-HjO -+ A-OH+B-H Sucrose

(Hydrolyzis Reactions] Lipases Sucrase
Proteases Glucose 4- Fructose
Nucleases

4 Lyases or Desmoiases Aldolase A-fi. -ju A = B+X-Y Histidine

(Group elimination to Decarboxylase I I i Histidine decarboxylase
form double bonds Fumarase X Y Histidine 4- C0?
without hydrolyzis} Citrate synthase

5. Isomerases Isomerase A-B->A-B Glucose-6-Phosphate

(Transfer of Groups Virta.se Illi X Isomerase
within a molecule} Epimerase ¥ X X Y Fruclose-6-Phosphaie

6. Ligases or Synthetases A+B+ATR -► Pyruvate + C0? + ATR

Synyhetases (Bond Carboxylases A-B4-ADP4-P1 i Pyruvate carboxylase
formation couples with Oxaloacetate 4- ADP + Pi
ATP hydrolyzis)
80 Chapter 2. Enzymes

Class 1 — Oxidoreductases
The enzymes of this class catalyze the redox reactions underlying biological
oxidation. The class has 22 subclasses. Coenzymes of this class arc NAD, NAD PH,
PAD, FMN, ubiquinone, glutathione, lipoicacid. Exam pics of subclasses arc enzymes
that act on the CH-OH group of donors, on the CH-CH group of donors, on the
CH-NH3 group of donors, on heme-containing donors.
The most common working names for oxidoreductases arc:
► dehydrogenases — oxidoreductases catalyzing the dehydrogenation of the
substrate using any molecules other than oxygen as the acceptor of hydrogen.
For example, the enzyme lactate dehydrogenase, which catalyzes the conversion
of lactate into pyruvate.

COO" Lactate COO"

1 dehydrogenase 1
HO-------C-------- H + NAD* ------------ C'------ O + NADH + H
1 1
ch3 ch3

| L-Lactate ] | Pyruvate J

► The enzyme succinate de hydrogenase catalyzes the conversion of succinate to

fumarate:

COO" COO-
Succinate 1
CH-. dehydrogenase
CH
| 2 + FADH + fadh,
CHj CH

COO- COO-

! Succinate j [ Fumarate ]

* reductases — if the transfer of hydrogen from the donor molecule is d i fTicuil L

to prove. Di hydrofolate reductase, or DHFR, is an enzyme that reduces
dihydrofolic acid! to tetra hydrofolic acid, using NADPH as electron donor:
► oxidases — oxidoreductases catalyzing the oxidation of substrates with molecular
oxygen as an electron acceptor without the inclusion of oxygen in the substrate
molecule;
► oxygenases — oxidoreductases catalyzing the introduction of one oxygen
atom into the molecule of the substrate with molecular oxygen as an oxygen
donor;
2.3. Enzymes Classification and Nomenclature

Xanthine oxidase

O
Z. IE

[~Xanitiinc ■ Uric acid 1

Carbon
monoxide
82 Chapter 2. Enzymes

► peroxidases -- oxidorcductascs catalyzing reactions with hydrogen peroxide as

an electron acceptor.

H2O2 + 2 H2O

Electron donor Oxidized electron donor

(e.g.. L-Ascorbic acid) (e.g., L-Dehydroascorbic acid)

Class 2 — Transferases
Transferases catalyze the transfer of various groups Prom one substrate (donor)
to another (acceptor), participate in the intcrconvcrsion reactions of various
substances, neutralization of natural and foreign compounds. Coenzymes arc
pyridoxal phosphate, coenzyme A. tetra hydro folic acid, methylcobalamin. The
class is divided into 9 subclasses depending on the structure of the transferred
groups. Examples of subclasses arc enzymes that carry one-carbon fragments,
aldehyde or keto-residues, acyl residues, nitrogen-containing groups, phosphorus-
containing groups.
An example of the transferases action is the reaction of transamination in the
synthesis of amino acids.

COO" COO"
Alanine
:oo" COO"
ansaminase
H3N+— C----- H c=o :=o + HJM*------C----- H
1 I

CH3 (CH^ c^3 (CH2)2

1
Alanine | Pyruvate
COO" COO"
Keloqlutarate | L'Glutamate |

Often the working name of transferase — kinase is used. These arc transferases
catalyzing the transfer of phosphate Prom ATP to a substrate (monosaccharides,
proteins, etc.), i.c.T phosphotransferase. For example, the fust step of glucose
activation in glycolysis is the conversion of D-glucose into glucose-6-phosphate.
2.3. Enzymes Classification and Nomenclature

Kinases arc the most important regulatory enzymes of the metabolic pathways.

Class 3 — Hydrolases
Hydrolases — enzymes that break intramolecular bonds in a substrate by attaching
elements of H,O, arc divided into 13 subclasses. Due to the complexity of many
substrates, a. number of enzymes retain trivial names, for example, pepsin, trypsin.
Coenzymes arc absent. Hydrolases arc widely represented by the gastrointestinal
tract enzymes (pepsin, trypsin, lipase, amylase, and others) and lysosomal enzymes.
They carry out the decay of macromolecules, forming easily adsorbed monomers.
Examples of subclasses arc groups of enzymes acting on esters, on ethers, on peptides,
on carbon-carbon bonds. Historically, the names of hydrolases evolved from the name
of the substrate with the end of the <-asc* — collagenase, amylase, lipase. DNA-asc.
The following working hydrolase names are most common:
► esterase - hydrolyzis of ester bonds. Cholesterol esterase catalyzes the hydro I yzis
of sterol esters into their component sterols and fatty acids;
84 Chapter 2. Enzymes

► lipases — hydroiyzis of neutral fats (triacylglyoerols). Lipases perform essential

roles in digestion, transport and processing of dietary lipids (c.g., triglycerides,
fats, oils) in most living organisms;
o
II
H2C —O—C—R H2C—OH H2C—OH
O
11
HC — O— C— R*
h2o^
O
fl
HC—O—C —
I
HC —O—C — R'
?
Lipase Lipase
O
|1
HaC—O—C—R"
O
II
h2c—0—c—
I
H5C—OH

[Triacyglyccrolj Diacyglyccrol | Monoacylglyccrol J

+

rafty acid

► phosphatase — hydroiyzis of phosphoric acid monocsters. Phosphatase enzymes

arc essential to many biological functions, because phosphorylation (c.g., by
protein kinases) and dephosphoryiation (by phosphatases) serve diverse roles in
cellular regulation and signaling;

O^Y^O
rRj
AR,°y“
r2
[Phosphatidate] 'Diacylglyccrol (DAG).
Diacylglycerol kinase

► glycosidases — hydrolyze O- and S-glycosidic bonds. Glycosidases catalyze the

hydroiyzis of glycosidic bonds in complex sugars. They arc extremely common
enzymes with roles in nature including degradation of biomass such asccllulo.se
(cellulase), hemicellulose, and starch (amylase);
2.3. Enzymes Classification and Nomenclature 85

► proteases, peptidases — hydrolyzis of proteins and peptides. Proteases arc

involved in digesting long protein chains into shorter fragments by splitting the
peptide bonds that link amino acid residues;
Hydrolysis sits for trypsin Hydrolysis site for tfirombin

Lysine or [ Arginine | [ Glycina ]

arginine

► nucleases — hyd rolyzis of nucleic acids. Nuclease is an enzyme capable ofcleaving

the phosphodicslcr bonds between monomers of nucleic acids. Nucleases
variously affect single and double stranded breaks in their target molecules. In
living organisms, they are essential machinery’ for many aspects of DN A repair.

OH

ho—P=O

O H(OH)

HO!— P=O

oI
o
86 Chapter 2. Enzymes

Class 4 — Lyases
Lyases arc enzymes catalyzing the cleavage of C-O, C-C, C-N, and other bonds,
as well as reversible cleavage reactions of various groups in a non-hydrolytic manner.
There are 7 subclasses. These reactions arc accompanied by the formation of a double
bond or the addition of groups to the double bond site. Lyases are complex enzymes.
Coenzymes arc pyridoxal phosphate, thiamine diphosphate, magnesium and cobalt
arc involved.
Enzymes arc divided into subclasses depending on the nature of the attacked
connection. Examples include enzymes acting on carbon-carbon bonds, carbon­
oxygen bonds, carbon-nitrogen bonds. For example, glutamate decarboxylase
converts glutamate to gamma-aminobutyric acid, the most important inhibitory
neurotransmitter of the central nervous system.

COO" NH3+

H3N+----- CH CH2
H+ CO2

ch2 A A. ch2

I
ch2
Glutamate
decarboxylase ch2

COO” COO”
[GABA]

Class 5 — Isomerases
Isomerases are enzymes that catalyze isomeric transformations within a single
molecule. Isomerases arc complex enzymes. Their coenzymes include pyridoxal
phosphate. deoxyad cnosyl cobala mi nT g I utat h ionc. monosaccharide phosphates
(glucose-1, 6-diphosphatc), etc. Isomerase subclasses arc distinguished depending
on the type of reaction. For example, racemases (reversible transformation of L-
and D-stcrcoisomers, epimerase (transformation of isomers with more than one
asymmetry center, for example, a-D-glucose into p-D-glucose), mutases (transfer
of chemical groups inside molecules). For example, phosphoglucomutasc converts
glucose-1.-phosphate to glucose-6-phosphate:

COO” COO”
Alanine
racemase
H3N+------ C------ H H------ C------ NH3+

I
ch3
I
ch3
[ L-Alanine | [p-Alanine]
2.3. Enzymes Classification and Nomenclature 37

[a-EXjIucose t-pbosphaie] (a-D-Glucose 6-phosphatc j

Class 6 — Ligases
Ligases (synthetases) arc enzymes that catalyze the attachment of two molecules
to each other using the energy of high-energy bonds of ATP (or other macroergs}.
Ligases arc complex enzymes. They contain nucleotide (UTP), biotin {vitamin HL
folic coenzymes. There arc 6 subclasses.
An example of subclasses is the group of enzymes by the type of the formed bond :
carbon-oxygen (C-O), carbon-sulfur (C-S), carbon-nitrogen (C-N), carbon­
carbon (C-C):
CQO

Glutamine h^n4----- c------ h

synthetase
+ ADP + Pj
CH,

I
CH,

Z\ Z\
I
o o o nh5
[L-Glulamate ] [ L-Glutamine ]

For example, DNA ligase is a specific type of enzyme that facilitates the joining of
DNA strands together by catalyzing the formation of a phosphodicstcrbond. h plays a
role in repairing single-strand breaks in duplex DNA in living organisms:
3'

DMA strand

UNA strand
I DMA strand
S3 Chapter 2. Enzymes

Review tests
1. Choose alt I he correct answers. Enzyme catalyzing Lhis reaction:

COO COO

HO—C—'H C=O
+ NAD +--------- ► I + NADH + H*
CH2 ch2
I
coo coo
f L-Malatej (jOxalgaceLate J

A. Belongs to the class of transferases.

B. Belongs to the class of oxidorediactascs.
C. Isa simple enzyme.
D. Is a holoenzyme.
2. Parkinson's disease is caused by a deficiency of dopamine in the substantia nigra of the
brain. Dopamine is produced in the brain front Ij-DOPA (L-di hydroxy phenylalanine)
by removal of a molecule of carbon dioxide. The L-DOPA is produced by oxidation of
tyrosine, which is in turn produced from phenylalanine by another oxidoreductase. The
enzyme that converts L-DOPA to dopamine Ls best classified as an/a:
A. Oxidorcductasc.
B. Transferase.
C. Hydrolase.
D. Lyase.
E. Isomerase.
Most synthetic reactions in which carbon dioxide is used require the vitamin biotin. The
biotin becomes covalently bound to a lysine molecule that is part of an enzyme, and the
biotin can then attach Io and carry the carbon dioxide molecule. Biotin is best referred
to as an/a:
A. Coenzyme.
B. Enzyme inhibitor.
C. Competitive inhibitor.
D. Organic acid.
E. Enzyme.
4. Mos! amino acids found in living things arc L-form amino acids. A few amino acids
found in antibiotics arc D-form. The enzyme that converts D-alanine Io L-aEanine is
best classified as an/a:
A. Transferase.
B. Hydrolase.
C. Lyase.
D. Isomerase.
E. Ligase.
2.3. Enzymes Classification and Nomenclature 89

5. Match the figure and the Idler:

2 2
Coenzyme NAD" composition:
A. Adenyl nucleotide.
B. Nicotinamide coenzyme.
C. Adenine.
D. Ribose.
E. Nicotinamide.
Coenzyme:
1. NAD \
2. FAD
3. Coenzyme A.
Vitamin:
A. Pantothenic acid.
B. B,
C. Niacin.
D. Biotin.
E. B,.
7. During replication, a number of DNA fragments are produced. Energy is required for an
enzyme to join these fragments together to form an intact strand of DNA. The enzyme
is classified as an/a:
A. Transferase.
B. Hydrolase.
C. Lyase.
D. Isomerase.
E. Ligase.
90 Chapter 2. Enzymes

8. What is the class of the enzyme catalyzing the reaction of glucose activation?

A. Oxidorcductascs.
B. Transferases.
C. Isomerases.
EX Hydrolases.
E. Lyases.

Situational Problems
1. With the prevalence in food of peeled cereals or bread made from high-grade flour,
hypovitaminosis B l may occur. Explain the rote that vitamin Bs plays in the body.
For this:
a) name the coenzyme which contains vitamin BL and enzymes, which require
this coenzyme to function:
b) write the process in which these enzymes arc involved and explain how the
process speed will change with a lack of B :
d) what disease develops in the absence of vitamin B,.
2. The patient has signs of pellagra, symmetrical dermatitis on the rear surface of the
hand, neck, face, stomatitis. The patient complains of nausea, abdominal pain,
diarrhea, lack of appetite, headaches, dizziness, depression.
a) what vitamin deficiencies cause these symptoms?
b) what coenzyme synthesis is reduced in this situation?

2.4. REGULATION OF ENZYMES AND METABOLIC PATHWAYS.

INHIBITION OF ENZYMES. DRUGS AS INHIBITORS OF ENZYMES
An important property of enzymes is the ability to be regulated by other molecules.
They can be active depending on changes in intracellular metabolism, and can also
respond to signals from the environment. Increasing or decreasing the speed of
individual reactions is done by changing the amount of the enzyme (it takes several
hours) or its activity (this happens very quickly). Both mechanisms arc often used.
There arc several ways to regulate the activity of enzymes (Fig. 2.18).

Availability of substrate or coenzyme

The rate of a chemical reaction is proportional to the product of the reacting
substances concent ration. Thus, a change in the amount of al least one of the substrates
2.4. Regulation of Enzymes ano MeiaDoiic Pathways. inniDilion of Enzymes...

Fig. 248. ways ol regulation of enzymatic activity

stops or starts (he reaction. An important parameter for the occurrence of enzymatic
reactions is also the presence ofcoenzymes necessary during the reaction. For example,
for the TCA cycle, this substrate isoxaloacctatc. The presence of oxaloacclate triggers
cycle reactions, which makes it possible to involve acctyl-CoA molecules in the
oxidation.

Genetic regulation
Genetic regulation (change in the amount of enzyme) can occur as a result of an
increase or decrease in its synthesis. From this point of view, enzymes can be divided
into several groups:
Constitutive — such enzymes that arc constantly formed in. the cell, regardless of
the presence of the substrate. These arc enzymes necessary to maintain cell activity,
for example, glycolysis enzymes, p-oxidation of fatly acids, DNA synthesis and repair.
Induced (adaptive) — the synthesis of these enzymes increases with the presence of
appropriate stimuli or inductors. For example, during pregnancy and after childbirth,
synthesis of the enzyme lactose synthase is induced under the influence of lactotropic
hormone, glucocorticoid hormones stimulate the synthesis of gluconeogenesis
enzymes, which ensures the stability of blood glucose concentration during prolonged
fasting and the central nervous system resistance to stress.
Repressed — the formation of such enzymes in. the cell, if necessary, is suppressed.
For example, in the liver, repression of the enzyme HMG-CoA reductase under the
influence of cholesterol. It affects the repression of gluconeogenesis enzyme synthesis
by insulin.
The change in the rate of enzyme synthesis (induction or repression) usually
depends on the amount of certain hormones or metabolites of the process.
92 Chapter 2. Enzymes

Com partmentalization
The compartmentalization is the accumulation of enzymes and their substrates
in one cell compartment (one organelle — endoplasmic reticulum, mitochondria,
nucleus, etc.). For example, the enzymes of the TCA cycle and [J-oxidation of fatly
acids arc located in the mitochondria, enzymes of protein synthesis on the ribosomes.
The regulation of enzyme activity is also carried out in different ways.

Covalent modification (phosphorylation/dephosph oryiat ion)

Regulation ofenzyme activity due to covalent modification occurs when attaching
or cleaving a certain group, most often phosphoric acid. Enzyme phosphorylation
occurson serine and tyrosine hydroxyl residues. The attachment, of phosphoric acid to
the protein is carried out by protein kinase enzymes, cleavage — protein phosphatase
(Fig. 2.19). This is a reversible form of regulation ofenzyme activity.

□
HO—1—•□*■
Target — Target
Vu Phosph Ofylated
UiiptiosphoryHtecl

Phosphatase

o
I1Q— h£o
L

Transferred | water ]
phosphate group

Fig. 2.19. Scheme of regulation of enzyme activity by phasphorylation/Hephosphorylation

2.4. Regulation of Enzymes and Metabolic Pathways, inhibition of Enzymes... 93

For example, in this way, glycogen phosphorylase and glycogen synthase enzymes
arc regulated in muscles. Du ringcxcrcisc, whileglycog.cn phosphoiwlascbecomcsactivcand
starts breaking down glycogen and burning glucose, during this lime glycogen synthase is
not active. When glycogen synthase active, glycogen phosphorylase — inactive (Fig. 2.20).
AcLivabon or glycogen phosphorylase and inaclivaLion
ot glycogen synthase by covalent modification

Glycogen phosphorylase - FJ Glycogen synthase - 1J

[active) V (maedwe)

Protein kinase A
ATI*

Glycogen phosphorylase
(inactive)
A Glycogen synthase
(active)

Fig. 2.20. Regulation of glycogen synthase and glycogen phosphorylase Dy covalent modification

Limited (partial) proteolysis

Regulation of enzyme activity by limited (partial) proteolysis of pro-enzymes
implies that the synthesis of a number of enzymes is carried out in the form of a larger
precursor (pro-enzyme) and; this enzyme is activated by cleaving one or more peptide
fragments from it. As a result of partial proteolysis (peptide cleavage), the primary
structure of the protein changes, the molecular weight changes, the conformation of
the enzyme and its active center changes (Fig. 2.21). This is an inc-vers iblc regulation of
the enzyme. In this way, proteolytic enzymes of the gastrointestinal tract (trypsinogen,
pepsinogen, pro-carboxypcptidasc) arc activated. The secretion ofenzymes outside the
cell in an inactive state allows you to protect cells from damage by digestive enzymes.
S^nal peptide

A-Jlocatatybc adivabor-
Activation
at low pH

Pepsin [

Fig. 221. The scheme of activation of pepsinogen and the formation of me active form of pepsin
94 Chapter 2. Enzymes

Protein-protein interactions
The term protein-protein interaction refers to a situation, where specific
metabolites, rather than metabolites of biochemical processes, act as a regulator. After
the influence of any factors on specific proteins, the activities of the sc proteins change,
and they, in turn, affect the given enzyme. Also, a change in catalytic activity can be
caused by the association or dissociation of the enzyme subunits. This is a reversible
form of regulation. An example of protein-protein interaction can be the regulation of
the activity of protein kinase A through the mechanism of association-dissociation.
Protein kinase A is a tetrameric enzyme consisting of 2 catalytic (C) and 2 regulatory
(R) subunits. Activator for protein kinase A is cAMP (cyclic AMP). The addition of
c.AM P to the regulatory subunits of the enzyme causes their release from the catalytic
subunits. Catalytic subunits are activated (Fig. 2.22).

Cid ic AMP

inactive
protein kinase A

Regulatory
subunits
Catalytic
subunits
Inactive catalytic
subunits

Fiql 222. Scheme of regulation of me activity of prole in kinase A using protein-protein interactions

Allosteric regulation
Allosteric enzymes arc built from two or more subunits: some subunits contain a
catalytic center, others have an allosteric center. Attaching the effector molecules to an
allosteric (regulatory) subunit changes the protein conformation and. accordingly, the
activity of the catalytic subunit. These effector molecules can function as activators or
inhibitors, meaning that the change to the protein will increase the rate of the reaction,
or prevent further binding of substrates at the active site, respectively. In short, an
activator will increase the reaction rate, while an inhibitor will decrease the rate or stop
it entirely (Fig. 2.23). A regulation of allosteric enzymes is reversible.
2.4. Regulation of Enzymes and MetaDolic Pathways, inhibition of Enzymes... 95

Active site (correct

conformation lor
substrate)

Substrate Active site (incorrect

conformation for
substrate]

tnnibitor site

activator Allosteric
inhibitor
Activator site

Fig. 223. Schematic representation of allosteric enzyme regulation

Regulation of the metabolic pathways

Enzymes work in all metabolic pathways of the cells and the body. Allosteric
enzymes usually stand at the beginning of metabolic paths, and their activity depends
on many subsequent reactions. Therefore, they are often key enzymes. The key
enzymes in the metabolic pathways usually catalyze:
i irreversible (-*) or partially reversible reactions:
► the slowest reactions;
* reactions at the beginning of lhe metabolic pathways or in branching places of
metabolic pathways.
The final metabolite of a biochemical process or the product of a given reaction
can be used as a negative or positive regulator. If the regulators arc the metabolite or
substrate of the reaction, then wc speak of direct regulation, it can be cither positive
or negative. The regulalorcan also be metabolites of biochemical pathways, somehow
related to this reaction.

Gene transcription I ■*"

'------------- ' Feedback inhibflton

Fig. 2.24. Scheme of regulation of enzyme activity by feedback inhibition

Tor example, the enzyme of the process of glycolysis, phosphofructokinasc, is

regulated by intermediate and final products of this decomposition. Moreover, ATP.
96 Chapter 2. Enzymes

citric acid, fructose-1,6-d ip hosphatc arc inhibitors, and fructose-2,6-bi.sphosphatc

and AMP arc activators of the enzyme (Fig. 2.25).

T njctosc-6-phosphate
Hatp

I L<ATP
{
H Citrate
HAMP y Fructose I I Phosphofructokinase
{-) Fructosc-2,6-bisP bisphosphatase
I *^ADP
T riKtosc-1,6-biphosphatc
(—) Phosphoeno Ip yru
(+} AMP
Fa 225. Scheme of glucose metabolism regulation
(+} Fructose-?,6-bisP

Inhibition of enzymes
Enzymes need to be tightly regulated to ensure that levels of the product do not rise
to undesired levels. This is accomplished by enzyme inhibition.
There arc two main directions of i nhibition. (Fig. 2.26):
► in terms of the strength of binding the enzyme to the inhibitor, inhibition is
reversible and irreversible;
► in relation to the inhibitor to the active center of the enzyme, the inhibition is
divided into competitive and noncompetitive.

Fa 2.26. Scheme of types of enzyme inhibition

Reversible inhibition
Reversible inhibitors bind to enzymes with non-covalent interactions such as
hydrogen bonds, hydrophobic interactions and ionic bonds. Multiple weak bonds
between the inhibitor and the active site combine to produce strong and specific
2.4. Regulation of Enzymes and Metaooiic Painways, inhibition of Enzymes... 97

binding, in contrast to substrates and irreversible inhibitors. reversible inhibitors

generally do not undergo chemical reactions when bound to the enzyme and can easily
be removed by dilution or dialysis.
Competitive inhibition — these inhibitors are structural analogs of substrates. They
bind in the active center of the enzyme, but cannot turn into a product. Reversible
competitive inhibitors (I) compete with the substrate (S) for the active site of the
enzyme (E) (Fig. 2.27).. With increasing, concentration of the substrate, it displaces
the inhibitor from the active site of the enzyme. For example, in reactions of the TCA
(tricarboxylic cycle, malonatc very similar in structure to succinate competes with
it for the active center of the enzyme succinate dehydrogenase, which catalyzes the
conversion of succinate to fumarate. The substrate and the inhibitor interact with the
same groups of the catalytic center of the enzyme. Malonatc can be displaced from the
active center of the enzyme by large concentrations of succinate.
Non-competitive inhibition — these inhibitors join the enzyme not in the active
center, but in another place, causing a change in the conformation of the enzyme and
its active center (Tig. 2.28). Therefore, reversible noncompetitive inhibition cannot
be eliminated by increasing the concentration of the substrate. The non-competitive
inhibitor can reversibly bind both the free enzyme (E) andl the enzyme-substrate
complex (ES). When the inhibitor concentration decreases, it dissociates from the
enzyme-inhibitor ( El) or cnzyme-substrate-inhibitor( ESl) complexes, and a gradual
restoration of the enzymatic activity occurs.

Fig. 227. Competitive inhibitor action

Normal

Fig. 228. Non-competitive inhibitor action

98 Chapter 2. Enzymes

Irreversible specific inhibitors — these inhibitors specifically bind or destroy

the functional group of the enzyme active center molecule, which is necessary for
the manifestation of its catalytic activity. Irreversible inhibition is different from
irreversible enzyme inactivation. Irreversible inhibitors arc generally specific for one
class of enzymes and do not inactivate all proteins: they do not function by destroying
protein structure but by specifically altering the active site of their target. Irreversible
inhibitors arc often poisonous. An example of such inhibition may be the action of
derivatives of fluorine phosphates. For example, diisopropyl lluorophosphatc is
a nerve poison because the enzyme acetylcholinesterase in. the synapses of neuron
has a reactive site serine. Diisopropyl fluorophosphatc transfers its phosphate to the
active site serine. The resulting phosphoenzyme is totally inactive. Consequently,
di isoprop vl fiuorophosphatc is a potent neurotoxin, with a. lethal dose of less than
100 mg (Fig. 2.29).

Diisopropyl phospnofkioridate
H
I
H3q..-C — GHa
0

Enzyme— Ser 195—O—C— R Enzyme — Ser 195— 0—P —. O

I
Acyl Enzyme I

1
C—CH3

H
Diisopropyl phosphoryl
Enzyme
(inactivated)

Ry. 229. Reaction of me irreversible iihibilor diisopropyl pnospnofluondate |DFP) with a serine protease

Irreversible nonspecific inhibitors — this type of inhibitors include hydrogen

sulfide, salts of lead, mercury, and silver. These substances bind to the protein part of
the enzyme outside the active center and form poorly soluble complexes. At the same
lime, the conformation of the whole enzyme molecule changes irreversibly and as a
consequence, changes of the active center occur.

Drugs as inhibitors of enzymes

Enzyme inhibitors arc found in nature and arc also designed and produced as
pan of pharmacology and biochemistry. Natural poisons arc often enzyme inhibitors
that have evolved to defend a plant or an imal against predators. These natural toxins
include some of the most poisonous compounds known. Artificial inhibitors arc
often used as drugs, but can also be insecticides such as malathion, herbicides such as
glyphosate, or disinfectants such as triclosan. Other artificial enzyme inhibitors block
acetylcholinesterase, an enzyme which breaks down acetylcholine, and arc used as
nerve agents in chemical warfare. Chemotherapy is the most common use for enzyme
2.4. Regulation of Enzymes ano MetaDoiic Pathways. inniDilion of Enzymes... gg

inhibitors as drugs to treat disease. Many of these inhibitors target a human enzyme
and aim to correct a pathological condition. However, not all drugs arc enzyme
inhibitors. Sonic, such as anti-epileptic drugs, alter enzyme activity by causing more
or less of the enzyme to be produced. These effects arc called enzyme induction and
inhibition and act through alterations of gene expression, which is unrelated to the
type of enzyme inhibition discussed here. Other drugs interact with cellular targets
that arc not enzymes, such as ion channels or membrane receptors.

Sulfonam ides as antibacterial substances

Sulfonamides or sulfa drugs arc competitive inhibitors and arc important in the
treatment of many microbial diseases. Sulfa drugs like sulfanilamide resemble the
p-ami.nobenzoate, a molecule used in. the formation of the coenzyme folic acid.
These analogues compete with metabolites in metabolic processes because of their
similarity but arc just different enough so that they cannot function normally in cellular
metabolism. The first anti metabolites to be used successfully as chemotherapeutic
agents were the sulfonamides, discovered by G. Domagk. Sulfonamides or sulfa drugs
are structurally related to sulfanilamide, an analog of p-aminobenzoic acid. The latter
substance is used in the synthesis of the cofactor folic acid. When sulfanilamide or
another sulfonamide enters a bacterial cell, it competes with p-aminobcnzoic acid
for the active site of an enzyme involved in the folic acid synthesis, and rhe folate
concentration decreases. The decline in folic acid is detrimental to the bacterium
because folic acid is essential to the synthesis of purines and pyrimidines, the bases used
in the construction of l)NA, RNA, and other important cell constituents. The resulting
inhibition of purine and pyrimidine synthesis leads to a cessation of bacterial growth or
death of the pathogen. Sulfonamides arc selectively toxic for many pathogens because
these bacteria manufacture their own folate and cannot effectively take up the cofactor.
In contrast, humans cannot synthesize folate and must obtain it in the diet: therefore,
sulfonamides will not affect the host.
O 0

Suffanitamide p-Aminobenzoic acid

J
100 Chapter 2. Enzymes

Treatment of methanol intoxication

A medical therapy based on competition at the active site is used to treat patients
who have ingested methanol, a solvent found in gas-line antifreeze. The liver enzyme
alcohol dehydrogenase converts methanol lo formaldehyde, which is damaging to
many tissues. Blindness is a. common result of methanol ingestion, because the eyes
are particularly sensitive to formaldehyde. Ethanol competes effectively with methanol
as an alternative substrate for alcohol dehydrogenase. The effect of ethanol is much
like that of a competitive inhibitor, with the distinction that ethanol is also a substrate
for alcohol dehydrogenase and its concentration will decrease over time as the enzyme
converts it to acetaldehyde. The therapy for methanol poisoning is a slow intravenous
infusion of ethanol, at a rate that maintains a controlled concentration in the blood
stream for several hours. This slows the formation of formaldehyde, lessening the
danger while the kidneys filter out the methanol to be excreted harmlessly in the urine.

Action nonsteroidal anti-inflammatory drugs

An example of a drug whose action is based on the irreversible inhibition of
enzymes is a widely used drug aspirin (acetylsalicylic acid}. Anti-inflammatory
nonsteroidal drug aspirin provides a pharmacological action by inhibiting the enzyme
cyclooxygenase, which catalyzes the reaction of the formation of prostaglandins from
arachidonic acid. As a result of the chemical reaction, the acetyl residue of aspirin
is attached to the free terminal OH group of serine cyclooxygenase. This causes a
decrease in the formation of the reaction products of prostaglandins, which have a
wide range of bio logical functions, including mediators of inflammation.

Anti-cancer drugs as inhibitors of enzymes

Another example of the structural similarity of some inhibitors to the substrates
of the enzymes is the drug methotrexate lo folic acid. Folic acid is a substrate of
dihydrofolatc reductase, an enzyme involved in making nucleotides that is potently
inhibited by methotrexate. Methotrexate blocks the action of dihydrofolatc reductase
and thereby halts the production of nucleotides. This block of nucleotide biosynthesis
is more toxic to rapidly growing cells than non-dividing cells, since a rapidly growing
cell has to cany out DMA replication; therefore, methotrexate is often used in cancer
chemotherapy.

25. CLINICAL APPLICATIONS OF ENZYMES. PLASMA PROTEINS

AND ENZYMES IN DIAGNOSIS OF DISEASES
Enzymes arc the preferred markers in various disease states such as myocardial
infarction, jaundice, pancreatitis, cancer, ncurodegenerativc disorders, etc. They
provide insight into the disease process by diagnosis, prognosis and assessment of’
response therapy. The use of enzymes in med icine occurs in the following areas:
► enzyme diagnostics;
► enzyme therapy;
► use of enzymes in medical technology and industry;
► use of enzyme inhibitors.
2.5. Clinical Applications of Enzymes. Plasma Proteins and Enzymes in Diagnosis of Diseases 1(H

Usage of isoenzymes for diagnosis of diseases

Enzymodiagnoslics is a study of the activity of enzymes of blood plasma, urine,
saliva in order to diagnose certain diseases. Isoenzymes are the enzymes, catalyzed the
same reactions, usually arc located in different tissues and differ in their properties.
Isozyme profiles arc often performed in. the clinical laboratory fordiagnostie purposes.
The definition of isozymes is often operational, i.c.. based on simple and reproducible
assay methods. Isoenzymes differ in:
* isoelectric point (charge isomers):
► molecular mass (size isomers):
► pH and temperature optima, heal lability;
► response to effectors;
► immunological behavior;
► pattern, of distribution in. different organs and cell components.
The table (Table 2.4) shows some scrum enzymes used in clinical diagnosis.

Table 2.4. Principle serum enzymes usefl in clinical diagnosis

Aminotransferases
Aspartate aminotransferase (AST, or SGOTl Myocardial infarction
Alanine aminotransferase (ALT, or SGOT) Viral hepatitis

Amytase Acute pancreatitis

Ceruloplasmin Hepalalepticular degeneration (Wil son's disease)

Creatine kinase Muscle disorders and myocardial infarction

y-Glutamyl transpeptidase Various liver diseases

Lactate dehydrogenase (isozymes) Myocardial infarction

Lipase Acute pancreatitis

Phosphatase, acid Metastatic carcinoma of tfie prostate

Phosphatase, alkaline (isozymes) Various bone disorders, obstructive liver diseases

One of the examples is lactate dehydrogenase (LDH) which catalyzes the reaction
of convening pyruvate into lactate:

0 OH
NADH •+ H
II OH
t NAD+ II n
HaC- -C| -v UH
II Lactate H II
0 Dehydrogenase o
Pyruvate Lactic Acid

This enzyme is composed of four subunits of two types: type M (specific for skeletal
muscle ) and type H (specific for heart). Different tissues produce different amounts
of the two subunits, which then combine to form five different tetramers (Fig. 230).
102 Chapter 2. Enzymes

LDH-1 LDH-2 LDH-3 LDH-4 LDH-5 LDHC

LDH
Tetramer 83 88 81 «
Subunit • o o
LDH-M LDH-H LDH-C
4
miniMrt
11 RM £l

DNA -------------------
1
Fi^ 2JO. Lactate dehydrogenase isofarms composition: LDffl (4H) - in the heart and in REC (red
blood cellsas well as the Dram LDH2 (3H1M} - in the reticuloendothelial system; LDH3 (2H2M) - in
lhe lungs; LDH4 (1H3M) - in the kidneys, placenta, and pancreas: LDH5 (4M) - specific for skeletal
muscle and liver

The activity of the enzyme in scrum blood of healthy people is low, because
the proteins can’t, penetrate through cell membrane to blood. If the membrane is
damaged, the enzymes leak to blood. The determination of tissue-specific enzymes
(or isoenzymes? activity in scrum blood allows us to diagnose different diseases, such
as myocardial infarction, hepatitis, and many others.

Densitometric patterns of LDH isozymes in normal and patient serum

SKirophweic analysis c<

LDH isozymes on gel

A B C
normal Acuw Aojie
serum Tyx&'Ua.- hepaaicis
irTaoion
e
LDH| -___ _

LDHs ------

LDHg

Fig. 2.31. Detection of different isoforrns of Isolate dehydrogenase in me serum of a healthy patient
a patient with myocardial infarction and hepatitis

On the other hand, diseases of various organs arc always accompanied by a specific
^enzymatic profile*. For example, myocardial infarction is accompanied by increased
activity in the blood of the isoenzymes: creatine phosphokinase (CK-M B). isoenzymes
lactate dehydrogenase I and 2 (LDH-1 and LDH-2), aspartate aminotransferase
(AST); troponin Tand I.
2.5. Clinical Applications of Enzymes. Plasma Proteins and Enzymes in Diagnosis of Diseases 103

Fig. 2.32. Enzymatic profile for detection of myocardial infarction

Enzymopathies
If the enzyme cannot perform its function, it is called cnzymopathology or
enzymopathy. Enzymopathies arc conditions associated with an abnormal increase
or decrease in the activity of enzymes. The most common decrease in their activity
is a violation of the relevant metabolic processes. With cnzymopathology, clinical
significance may be:
► accumulation of subsume reaction. For example, phenylalanine in
phenylketonuria, free bilirubin in physiological jaundice of the newborn, some
fats in diseases of lysosomal accumulation (lipidosis):
► product deficiency. For example, melanin in albinism, catecholamines in
parkinsonism;
► both features simultaneously, as in glycogenosis, accompanied bv hypoglycemia
with an excess of glycogen in the liver:
By the nature of the violation, primary and secondary enzymopathies arc
distinguished. Lack of vitamins and their coenzyme forms is also the cause of acquired
enzymopathies.

Use of enzymes as a medicine

Enzymothcrapy is the use of enzymes as a medicine in replacement therapy
of diseases. For example, the most common enzyme preparations arc complexes
of enzymes of the gastrointestinal tract, containing pepsin, trypsin, amylase,
etc., and used for substitution therapy for disorders of digestion of substances in
the gastrointestinal tract.
Tissue enzyme hyaluronidase is needed by the body to reversibly change the
permeability of the intercellular substance, which is based on hyaluronic acid. The
104 Chapter 2. Enzymes

dosage form of hyaluronidase is administered to soften scars, the appearance of

mobility in the joints, the resorption of hematomas.

Use of enzyme inhibitors in medical treatments

Enzyme inhibitors arc widely used in the treatment of diseases as medicines, Al
present, protease inhibitors (kontrykal, gordox) arc widely used in pancreatitis —
conditions when the activation of digestive enzymes occurs in ducts and pancreatic
cells.
Cholinesterase inhibitors (prozerin) lead to the accumulation of the
neurotransmitter acetylcholine al synapses and are indicated for myasthenia, motor
and sensory disorders in neuritis, radiculitis, and psychogenic impotence.
Preparations containing monoamine oxidase inhibitors increase the production of
catecholamine neurotransmitters in the CNS in the treatment of Parkinson’s disease.
Suppression of the activity of monoamine oxidase (destroying catecholamines)
maintains normal signaling in the nervous system and is used for depression treatment.
An giotens in-converting enzyme inhibitors (captopril, enalapril, etc.) arc used as
an antihypertensive agent and cause the expansion of peripheral vessels, reducing the
load on the myocardium, reducing blood pressure.
Allopurinol is an inhibitor of xanthine oxidase, an enzyme for catabolism of
purines, is required to reduce the formation of uric acid and suppress the development
of hyperuricemia and gout.
HMG-CoA reductase inhibitors (lovastatin, lluvastatin, atorvasLalin) arc used to
reduce the synthesis of cholesterol in atherosclerosis, diseases of the cardiovascular
system, and dyslipoprotcinemia.

The use of enzymes in medical technology

The specificity of enzymes to certain substrates is widely used in laboratory
diagnostics.
Many laboratory1 methods arc based on the interaction of an externally added
enzyme with the compound to be determined. For example, glucose oxidase is used
for detection of amount of glucose in blood and urea samples.
Enzyme immunoassay’s based on the formation of the enzyme-antigen-antibody
ternary complex. The substance to be determined is not an enzyme substrate, but an
antigen. The enzyme can attach this antigen near the active site. If there is an antigen
in the medium, then when adding antibodies and forming a ternary complex, the
activity of the enzyme changes. Enzyme activity is measured by any method.

Review tests
1. In a patient's blood, the activities of lactate dehydrogenase (LDII4. LDII 5), alanine
aminotransferase. carbamoyl ornithine transferase are increased. What organ is affected
by the pathological process?
A. In skeletal muscles.
B. In the myocardium (myocardial infarction is possible).
C. In the liver (hepatitis is possible).
D. In kidneys.
E. In connective tissue.
2.5. Clinical Applications of Enzymes. Plasma Proteins and Enzymes in Diagnosis of Diseases 105

2. Match the figure and the letter.

Medicine substance:
1. Prozcrin.
2. Theophylline.
3. Aspirin.
Mechanism of action:
A. Structural analogue of para-aminobenzoic acid.
B. Phosphodiesterase inhibitor
C. Xanthine oxidase inhibitor.
D. Structural analogue of acetylcholine.
E. An irreversible inhibitor.
3. Choose the correct answer. Most growing cells require folic acid. Bacteria synthesize
their folic acid from a number of materials, including para-aminobenzoic acid. We slow
bacterial growth by treating infections in humans with sulfa drugs. (The sulfa drugs are
very similar to para-aminobcnzoic acid and act as competitive inhibitors, keeping the
bacteria from synthesizing folic acid.) If the sulfa drugs kill bacteria, why don't they
harm the human too?
A. There are Loo many bacteria, and some consume the sulfa drugs and die, leaving
the remaining bacteria to overwhelm the host.
B. Humans don’t make folic acid (it!s a vitamin for us) anyway, so the sulfa drugs
usually don! harm humans.
C. Humans don't metabolize folic acid.
D. Humans don’t metabolize sulla drugs.
E. H umans can take extra folic acid to compensate for the sulfa drugs.
4. Methanal is a very poisonous substance, accounting for a number of deaths and many
cases of blindness yearly. While the methanol itself is sometimes not toxic, it may be
metabolized by the liver to more toxic molecules such as formic acid or formaldehyde.
Before more efficacious treatments were developed, a person who had ingested methanol
was often given a large amount of ethanol orally. T he purpose of this treatment was:
A. To allow5 the ethanol to act as a competitive inhibitor of the methanol metabolism.
B. To allow the ethanol to act as a noncompetitive inhibitor of the methanol
metabolism.
C. To make the patient forget about the pain so that the operation could proceed.
D. To deaden the tissues to pain.
E. To dissolve the methanol and move it through the circulation faster.
5. Match the figure and the letter. The effect of competitive inhibition on the speed of the
enzymatic reaction.
106 Chapter 2. Enzymes

Chart Analysis:
A. Curve with inhibitor.
B. Km with an inhibitor.
C. Initial speed.
D. Km. without inhibitor.
E. Curve without inhibitor.
6. A 42-year man suffering from gout has increased level of uric acid in the blood.
Allopurinol was prescribed to decrease lhe level of uric acid. Competitive inhibitor of
what enzyme is allopurinol?
A. Xanthine oxidase.
B. Adenosine deaminase.
C. Adenine phosphoribosyltransrerasc.
D. Hypoxantin phosphoribosyl transferase.
E. Guanine deaminase.
7. Match the figure and the letter.
The mechanism of regulatio n of enzyme activity:
1. Allosteric regulation.
2. Ph osph.oryialion - dephosp horyliatio n.
3. Protein-protein interactions.
Features of regulation:
A. There is a change in the primary structure of the enzyme.
B. Inhibition is irreversible.
C. Is carried out by activating or inactivating protein kinases and protein phosphatases.
I>. Is accompanied by dissociation or association of protomers or protein complexes.
8. A 47-year-old patient was brought to an emergency department with the diagnosis of
myocardial infarction. What lactate dehydrogenase (LDI1) fraction's activity would
prevail in the patient s blood serum during lhe first two days after hospitalization?
A LDH4.
B. LDH6.
C. LDH3.
D. LDH1.
E. LI)H5.

Situational Problems
1. Pepsinogen (an inactive form of the enzyme) that forms in the main cells of the
stomach has a molecular weight of 42.000 D. I n the gastric juice, the pepsinogen
turns into lhe active enzyme pepsin, while its molecular weight decreases to
35.000 D. Explain the mechanism of regulation of enzyme activity. To do this:
a) draw a scheme for regulating the activation of pepsin:
b) name the class of the enzyme that converts pepsinogen to pepsin:
c) indicate which levels of protein structural organization change when pepsin
is activated.
2. The interaction of the neurotransmitter acetylcholine with M-cholinergic receptors
causes a smooth muscles of lhe internal organs to contract — the intestines,
stomach, gall and bladder, bronchi, as well as constriction of (he pupils. In addition,
M-cholinergic receptors are found in the central nervous system. Atropine is a
2.5. Clinical Applications of Enzymes. Plasma Proteins and Enzymes in Diagnosis of Diseases 107

drug used in clinical practice Io relieve smooth muscle spasms and belongs to the
group ofanlispasmodics. What is the mechanism of action of this drug?
a) comparison with neurotransmitters and drugs, detection of similar functional
groups;
b) suggest the mechanism of action of atropine as a drug that relieves smooth
muscle spasms:
c) explain why an overdose of atropine can cause motor and mental agitation.
 Chapter 3
GENE EXPRESSION AND PROTEIN
SYNTHESIS

3.1 Nucleic Acid Structure

32. ONA Replication
3.3. DNA Damage & Repair
3.4. Transcription: RNA Synthesis
3.5. Genetic Code Properties
3.6. Translation: Protein Synthesis
3.7. Protein Posttranslational Modifications
3.8. DNA, RNAand Protein Synthesis Inhibitors
3.9. The Regulation of Gene Expression
3.10. The Effects of Mutations
3.11 Recombinant DNA Technology

Nucleic acids are an important class of macromolecules found in all cells and
viruses. Nucleic acids arc responsible for the storage and expression of genetic
information. The three main processes used by ail cells to maintain and propagate
their genetic information are replication, transcription, and translation.

3X NUCLEIC ACID STRUCTURE

There are two types of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA). Although their name suggests that their location is in the cell nucleus,
some nucleic acids arc also present in the cytoplasm and mitochondria. Nucleic acids
are present in most living cells and represent hereditary determinants of all living
organisms.
Nucleic acids are polymers of deoxy ribonucleotides or ribonucleotides. All
nucleotides have a common structure: a phosphate group linked by a phosphodicstcr
bond to a pentose that, in. turn, is linked to a nitrogenous base (Fig. 3.1). The
nitrogenous bases fall into two types. Purines have a double-ringed structures
(Fig. 3.2). Pyrimidines have a single-ringed structures {Fig. 3.2).
The same two purines are present in. both DNA and RNA. The pyrimidines in
DNA arc cytosine and thymine, whereas, in RNA. uracil is present instead of thy mine.
The only difference between thymine and uracil is the absence of a methyl group at
position C5 (Fig. 3.2).
Two types of pentose sugars arc found in nucleic acids (Fig. 3.3). In DNA, the
pentose is deoxyribose. In RNA. the pentose is ribose. Deoxyribose is similar in
structure to ribose, but it has an II instead of an OH al the 2d carbon atom (Fig. 3.3).
3.1. Nucleic Acid Structure 109

Nitrogenous base

Fig. 3.1. A simple diagram of a nucleotide. Eacn nucleotide is composed of three parts: a 5-carbon
sugar, a phosphate group, and a nitrogenous base. Bom the phosphate group and nitrogenous base
are attached to the central pentose sugar: me nitrogenous base is attached to me T carbon atom via a
glycosidie bond ano the phosphate groups are covalently linked to me 5' carbon atom

Fig. 3.2. Nitrogenous bases molecular structures. The base is attached to a ribose or deoxyribose
molecule through a bond to me nitrogen atom shewn in color

[ [W-2-Deoxynbcise]

Fig. 3.3. Ribose and deoxyribose sugars

A nucleoside consists of a nitrogenous base covalently attached to a sugar ( ribose

or deoxyribose) but without a phosphate group. When a phosphate group is added, the
basc-sugar-phosphalc is called a nucleotide (Table 3.1).
110 Chapter 3. Sene expression and protein synthesis

Table 3.1. Name of me bases and their corresponding nucleosides and nucleotides

RNA

Adenine (A) Adenosine (A} Adenosine 5'-monophosphate (AMP)

Guanine (G) Guanosine (G) Guanosine 5’-monophosphate ^GMP)

Cytosine (C) Cytidine (C) Cytidine 5-monophosphate (CMP)

Uracil (UJ Uridine (U) Uridine S'-monophosphare (UMP)

DNA

Adenine (A) Deoxyadenosine (A) Deoxyadenosine 5 -monophosphate (dAMP)

Guanine (G) Deoxyguanosine (G} Deoxyguanosine 5'-mono phosphate (dGhlP)

Cytosine (C) Deoxycytidine (C) Cleoxycyl>dine 5' - monophosphate (dCMP}

Thymine (T) Deoxythymidine (T) Deoxythymidine S '-monophosphate idTMP)

Nucleotides arc linked into a polynucleotide chain by a phosphodiestcr bond that

connects the 3’ carbon atom of one nucleotide and' the 5' carbon atom of another
(Fig. 3.4) forming the phosphate-sugar backbone from which, the nitrogenous bases
protrude. The sugar of the terminal nucleotide at one end of the chain has a free 5’
phosphate group. The sugar of the terminal nucleotide al the other end has a free 31
hydroxyl group.
[n nucleic acid synthesis nucleoside triphosphate, NTP.. is a precursor: however,
nucleoside monophosphate, NMP, is directly incorporated to the polynucleotide
growing chain and two terminal phosphate groups of the NTP arc released in the form
of pyrophosphate (Fig. 3.5). Nucleotides arc added to a new strand of DNA or RNA
by extending the 3’ end of the chain.
DNA molecule consists of two strands that coil around each other to form
a double helix like a twisted ladder. The two strands of the helix run in opposite
directions, meaning that the S’-end of one strand is paired up with the 31~end of
its matching strand (Fig. 3.6 A). One strand is held to another by hydrogen bonds
between the bases. The sequencing of this bonding is specific: adenine bonds only
with thymine, and cytosine only with guanine. Adenine and thymine form two
hydrogen bonds between them, cytosine and guan inc form three hydrogen bonds
between them (Fig. 3.6 13). More simply, C bonds with G and A bonds with T. Il's
called complementary base pairing because each base can only bond with a specific
base partner.
3.1. Nucleic Acid Structure 111

Fig. 3.4. A structure of a polynucleotide chain Sugars are joined together Dy pnosphate groups
via pnosphodiester bonds Del ween me third and fifth carbon atoms of adjacent sugar rings. The
phospnodiester bond is indicated by red arrows. The prime or {*) designation is used when me sugar
is attached to me base to distinguish me base atoms from the sugar atoms
 Guanosine triphosphate degxyribo nucleotide (dQTP)
t------------------------------------------------------- X------------------------------------------------------- V

Guanine nueleotids <ClGMP)

Chapter 3. Sene expression and protein synthesis

Deoxy ribose

Diphosphate released,
energy used for synthesis

i rip hosp hate

Longer DNA strand Longer DNA strand
nucleotide

Fig. 3.5. Nucleic acid synthesis. (A) NTPJS are the precursors used In DNA synthesis. A nuclei tide formed from phosphoric
acldr deoxyribose, and an erganIc base, guanine. <B) NMP moiety of NfTP is added to the3!-0H end of a polynucleotide chain
3.1. Nucleic Acid Structure 113

Sugar-phosphaSe
bacWxcte

Phosphorus

Carbon in

,34 nm “backbone7

3.4 nm

Bases

2 nm

Fig. 36 The 3D double helix structure ol DMA. Complemenlary base pairings are responsible for the
double-helix structure of DNA. f A) The structure of a douflle-stranded ONA. The two strands of ONA
run in opposite directions to each otner and are thus anti parade I. (B) Hydrogen bonding between
complementary base pairs. Adenine and thymine form two hydrogen bonds, and cytosine and
guanine form three hydrogen bonds

RNA molecule is quite similar to DNA. However, whereas DNA molecules arc
usually long and double-stranded, RM A molecules arc much shorter and single­
stranded. RNA molecules play many critical roles in the cell but arc mainly involved
in protein synthesis (translation) and its regulation.
One of the key features of any RNA is that it can fold upon, itself via base pairs
between complementary nucleotides to form a three-dimensional structure (Fig. .3.7).
The secondary structure of RNA can be predicted by experimental data on the
secondary structure elements.
114 Chapter 3. Sene expression and protein syiitnesis

Stem Hairpin loop

I
o—f

A Bulge loop

20 GUCC T
i
GCGCGGUG CACCUGA U-4O
I I. - I I I * I I I I I I I t
G UGG A C U

Fig. 3.7. Different types of RNA secondary structures. (A) The first structure (upper row, from [eft to
right) represents a stem with six paired Dases. Tne second structure represents a nairpin loop having
three paired and seven unpaired Dases. Tne tnird structure is a pseudoknot which is one of the most
common type in whicn tne unpaired base of two hairpins structure get paired with tne Diner one. Tne
forth structure (bottom row, from left to right) represents a bulge loop. The fiftn structure represents
an internal loop. Tne sixth structure represents a brancn loop, a multi-loop tn at contains al least three
branches. (B) RNA secondary structure of Estf?eriitt?ra 5S rRNA
3.1. Nucleic Acid Structure 115

The Ihree major types of RNA include: messenger RNA (mRNA), ribosomal
RIMA (rRNAh transfer RNA ([RNA). These RNAs participate in protein synthesis
(Table 3.2).
Messenger RNA (m RNA) carries the message from the DNA, which controls most
cellular activities in a cell. mRN.A accounts for just 5% of the lota! RNA in a cell.
mRN.A transcripts contain the sequence information that ribosomes need to translate
io prole in. mRN.A is relatively short-lived in a cell.
Eukaryotes have devised modifications at both termini to prevent mRNAs from
exonuclease degradation from both 5’- and 3’-cnds and, thus, stabilize the mRNA
(discussed below).
Transfer RNA (tRNA) is the smallest of the three types of RNA. It acts as an
adapter in. the translation of mRNA into proteins. The main function is the transfer of
amino acids during protein synthesis. Each of the 20 amino acids has a specific tRNA
that binds with it and transfers it to the growing polypeptide chain. All tRNAs have
certain common features, including the secondary and tertiary structures (Fig. 3.8).

Fig. 3.8. The tRNA structure. The canonical tRNA (left) secondary structure has a clover-leaf shape.
In me celL it folds into a compact L shape (right). tRNA mole cutes fold into a cloverleaf structure with
four key regions. The acceptor stem (3'-CCA) carries an amino acid; the anticodon associates wim the
mRNA codon (via complementary base pairing): the T arm associates with the ribosome (via the E, P
and A binding sites}; me D arm associates with me tRNA activating enzyme (responsible for adding the
amino acid to me acceptor stem) •; discussed in Ch. 3.5r 3.6)

Interestingly, tRNAs have the numerous numbers of post-transcriptional

modifications among all RNAs, e.g., methylation. These modifications concentrate
in the anticodon loop and the tRNA core region, where the I)- and T-loop interact
116 Chapter 3. Sene expression and protein synthesis

with each other, stabilizing the overall structure of the molecule. Three adjacent
nucleotides constitute a unit known as the codon, which codes for an amino acid,
whereas an anticodon is a sequence of nucleotides that is complimentary to codon
(discussed in Ch. 3.5). Importantly, tRNAs have two properties:
> it represents a single amino acid, to which it covalently attached;
► each tRNA contains a set of three nucleotides called an anticodon. The
anticodon of a given tRNA can bind lo one specific mRNA codons (Fig. 3.9).
The anticodon enables the tRNA to recognize the codon via complementary
pairing.

3*
Amino acid
attachment sits

tRNA

AniicoOon kx»p

Anticodon

cue
LLL
mRNA

Fig. 3.9. Pairing of me tRNA anticodon with me mRNA codon proceeds from me S' end of the codon

The ribosome (Fig. 3.10) is an organelle that is present in large numbers in all
living cells and serves as the site of protein synthesis. Ribosomal RNA (rRNA) is
the major structural component of the ribosome and accounts for 80% of the total
RNA present in the cell. rRNAs perform critical functions in the ribosome that a How-
protein synthesis to occur: they arc involved in binding to mRNA, recruiting tRNA,
catalyzing the formation of a peptide bond between two amino acids.
Cytoplasmic ribosomes in eukaryotes contain four types of rRNA molecules,
whereas prokaryotic ribosomes contain only three types of rRNA molecules (Fig. 3.10).
3.2. DNA Replication 117

Mammalian mitochondrial ribosomes have small 28S and large 39S subunits,
together forming a 55S ribosome. Mitochondrial ribosomes have only two
mitochondrial, IlSand I6S, rRNA molecules.

Prokaryotic ribosome Eukaryotic ribosome

70S SOS
SOS
subunit 60S
subunit

90S 40S
subunit subunit

Fig. 3.1 G. Tne ribosome is a comp tex subcellular structure or particle made of rRNA molecutes and
proteins trial form a factory for protein synthesis in cells. Prokaryotes nave 70S ribosomes respectively
suDanils comprising the small subunit of 30S and tne large subunit of 50S. Eukaryotes nave SOS
ribosomes respectively comprising of tne small AOS ano tne large 60S subunits. Su Du nils are named
according to their rate of sedimentation, measured in SvedDerg units [ST SuDunits are composed
of numerous rRNA and protein molecules: SOS: 5S RNA, 23S RNA. 34 proleins; 30 S: 16s RNA,
21 proteins; 60S: 5S RNA, 28S RNA, 5.8 RNA, about 49 proteins, 40S: 18S RNA, about 33 proteins

Table 32. Three main types of RNA molecules: structure and function

Structure Short, unstable, Stable RNA molecule Short (70-90 nucleotides),

singfe-stranded RNA composing 60% of ribosome's stable RNA with extensive
corresponding to a gene mass intramolecular base pairing:
encoded within DNA contains an amino acid
binding site and an mRNA
binding site

Function Serves as an intermediary Ensures the proper alignment Transports the correct
between DNA and protein: of mRNA tRNA. and ribosome amino acid to the site of
used by ribosome to direct during protein synthesis; protein synthesis in the
synthesis of protein it catalyzes peptide bond ribosome
encodes formation between amino acids

32. DNA REPLICATION

I t is crucial that the genetic material is reproduced accurately. During reproduction,
DNA is replicated and passed from one generation of cells to the next and ultimately,
from parent organisms to their offspring. DNA replication is the biological process
that occurs in all living organisms.
118 Chapter 3. Sene expression and protein syntnesis

Semi-Conservative Mechanism of DNA Replication

The DNA strand that is copied to form a new strand is called a template. The
mechanism is semi-conservative: during DNA replication, both strands of the double
helix act as templates for the formation of new daughter DNA molecules (Fig. 3.11).
The parental chain dictates the sequence of the daughter chain. DNA synthesis
proceeds in the direction: from the 5' (phosphate) end to the 3' (hydroxyl) end. DNA
replication occurs only in. S-phase of the cell cycle.
A
T A T A T A

A T A T A I

G C G C G C

C G C G C G

T A T A I A

T A
T A T

T A
+ A T

T A
A T A T A T

G C G C G C

G C G C G C

C G C G C G

Cwuains ore parent strand Corus-ns second patent strand

Parent
and one newly aynittesized and one newly synthesized
strand coTplememai-y strand compaemeniay strand:

Repicon 1 fleplfcon 2 Replioon 3

Orqin 3

Black represents- parental DNA siranefe

Green represents newly synthesized DNA strands

Fig. 3.11. DNfl replication.. (A) semi-conservative replication: earn strand in the double helix acts as
a template for synthesis of a new, complementary strand. The result is two DNA molecules with one
original strand and one new strand. (B) part of a eukaryote chromosome showing multiple Origins
(1.2, 3) of replication, each defining a repficon (1, 2,3}. Replication may start al different times in
S-prtase. Here 1 and 2 begin first, men 3. as me replication forks proceed bidirectionally (indicated
by a Diack arrows}, mey create replication bubbles (indicated by a red arrow] mat meet and form larger
bubbles. Tbe end result is two semi-conservatively replicated duplex DNA strands
3.2. DNA Replication 119

DNA Replication: DNA Dependent DNA Polymerase

DMA replication employs a large number of proteins and enzymes, each of which
plays a pivotal role during the process (Table 3.3). The key enzyme is DNA dependent
DNA polymerase, also known as DNA pol, which adds nucleotides one by one to
the growing DNA chain that arc complementary to the template strand. Eukaryotic
cells contain five DNA polymerases: a, p, y, ft. and e. Polymerase y is located in
mitochondria and is responsible for the replication of mitochondrial DNA. The other
four enzymes arc located in the nucleus and arc involved in nuclear DNA replication.
Polymerases a, fi, and e arc most active in dividing cells. In contrast, polymerase 0 is
active in both nondividing and dividing cells (Table .3.4).
Table 3.3. Core proteins al the replication fork

Topoisomerases Prevents torsion by DNA breaks

Helicases Separates 2 strands
Primase RNA primer synthesis
Single strand binding proteins Prevent reannealing of single strands
D NA polymerase Synthesis of new strand

Table 34. A current view: roles of subunits of replicative DNA polymerases

Chromosomal repl leases 8 and £

Proofreading subunit 8 and e
Primase a
Primer extension u
Leading-strand Pol £
Lagging-strand Pol 8
Okazaki fragment processing 8
Repair of DNA damage
Mitochondrial replicase
P
T

DNA Replication: Replication Fork

Rapid and faithful replication of chromosomal DNA is executed by large
multiprotein complexes called chromosomal repl leases. The replication begins with
creation of a growing replication, fork by helicase that separates the DNA strands
al the origin of replication (Tabic 3.5, Pig. 3J4). As the result, the DNA lends to
become more highly coiled ahead of the replication fork. Topoisomerase breaksand
reforms DNA’s phosphate backbone ahead of the replication fork, thereby relieving
the pressure that results from this supcrcoiling. Once the helix unwinds, the single
strands of DNA arc bound by single-strand DNA binding protein (SSB). Primase
then synthesizes a short piece of RNA (5-IO nucleotides long), called a primer,
complementary-’ to the template strand that provides a 3’-end for DNA polymerase
to use for DNA synthesis. Primers arc required because DNA pol can only add
nucleotides to the existing nucleotide chain and cannot begin synthesis de novo.
120 Chapter 3. Sene expression and protein synthesis

Table 3.5. Steps in ONA replication

1 Enzyme helicase unwinds the parental double helix DMA

2 The srngfe-strand binding proteins stabilize the DNA

3 The leading DMA strand is synthesized continuously in the 5' to 3" direction by DMA pot

4 The tagging strand is synthesized discontinuously via Okazaki fragments

5 RNA primers are removed, gaps are filled by DNA pol. then DNA ligase joins the Okazaki fragments

Each primer, bound to its complementary DNA strand, is elongated by DNA pol,
thereby synthesizing a now daughter strand. Importantly, DNA pol adds nucleotides
to 3’-end of the primer; therefore, DNA synthesis always proceeds in a 5’ to 31
direction. As shown in Fig. 3.12, synthesis of one daughter strand, called the leading
strand, proceeds continuously from a single RNA primer in the 5’ to 31 direction, in
the same direction as the movement of the replication fork. Synthesis of the other
daughter strand, called the lagging strand, requires a slight delay before undergoing
replication and occurs discontinuously via Okazaki fragment in the direction opposite
to the direction in which the replication fork is moving. The lengths of Okazaki
fragments are generally between 100 to 200 nucleotides long in eukaryotes.

Single-strand binding
The leading strand ts
Helicases unwind the synthesized continuouBly
proteins stabilize the
parental double helix. in me 5— 3’ direction by
unwound parental DNA.
DMA polymerase.

DNA polymerase

Replication fork
The lagging strand is
RNA primer
synthesized discontinuously.
Primase Okazaki fragment Primase synthesizes a short
being made RNA primer, which is
extended by DNA polymefase
DNA---------- Id form an Okazaki fragment
polymerase
Parental DNA

.After the RNA primer is

replaced by DNA ^by another
DNA potymerase, hot shown),
DNA tiga&e joins me Okazaki DNA Eigase
fragment to me growing strand.

Overall direction of replication

Fig. 3.12. The DNA replication scheme. The helicase unzips the double-stranded DNA for replication,
making a forked structure. The primase synmesizes RNA primers that bind to the single-stranded
DNA to initiate ONA synthesis by the DMA pol. DNA pol synmetizes only in the 5' to 3* direction, so
it replicates the leading strand continuously. Synthesis of me lagging-strand is discontinuous via
Okazaki fragments
3.3. ONA Damage & Repair 121

The energy required to drive the reaction comes from cutting high energy
phosphate bonds on the NT PS usedi as the source of the nucleotides needed in the
reaction (Fig. 3.5).
The RNA primers of Okazaki fragments arc subsequently degraded and the gaps
are tilted by DNA pol and sealed by DMA ligase (Fig. 3.I2). The ONA ligase is an
enzyme that creates phosphod tester bonds between adjacent nucleotides between
Okazaki fragments.

DNA Replication: Origin of Replication

The origin of replication is sequence in a genome DNA at which replication is
initiated. DNA replication origins arc typically rich in A-T base pairs, which are held
together by only two hydrogen bonds. Eukaryotic DNA replication is initialed by
forming many replication forks al multiple origins to complete DNA duplication
of >3 billion base pairs (bp) of DNA in the human genome in the lime available
during the S phase of a cell cycle. The location and distribution of replication
origins throughout the genome define rcplicons, which arc large sequence domains
copied by the bidirectional movement of the replication fork away from an origin.
Depending on. cell type, observed rep I icon sizes ranged from 30 to 450 kb.
DNA replication starts with the binding of proteins to the origin, of replication,
opening up a replication bubble in the DNA (Fig. 3.11 B). Each end of the bubble is
a replication fork. DNA replication occurs in both, directions. As replication nears
completion, bubbles of newly replicated DNA meet and fuse, finally forming two new
molecules.

33. DNA DAMAGE & REPAIR

Mechanisms for Maintaining DNA Integrity

The DNA sequence can be altered as the result of copying errors introduced by
DNA pol during replication or by environmental agents (Fig, 3.I3). DNA damage is
an alteration in the chemical structure of DNA, such as a break in a strand of DNA
or a double-strand breaks in DNA, a base missing from the backbone of DNA. or a
chemically changed base.
The accuracy of DNA replication is critical to cell reproduction. During replication,
DNA. pol must select the correct deoxynucleotide Irom a pool of structurally similar
nucleotides to preserve the complementary base pairings of DNA. Errors during DNA
synthesis arc rare events: however they can. have serious consequences that arc central
to the etiology of diseases and aging. Some of the errors arc corrected immediately
during replication through, proofreading by DNA pol, and some arc corrected after
replication through a post-replicative mismatch repair. If the damage cannot be
repaired, the cell can undergo programmed cell death (apoptosis) to avoid passing on
the defective DNA. If these mechanisms fail, mutations are passed down.
DNA can be damaged via environmental factors as well. The damage can be
caused by UV or X rays or genotoxic chemicals. UV can result in a covalent, linkage
between two adjacent pyrimidine bases in DNA, creating pyrimidine dimers that
122 Chapter 3. Sene expression and protein synthesis

prevent DNA replication. Ionizing radiation and certain drugs can block replication
by creating double-strand breaks in the DNA. Intercalating agents, c..g., ethidium
bromide, can cause abnormal insertions and deletions in the DNA sequence.

DMA Damage

F^ 111 DNA damage can occur naturally or via environmental factors

DNA damage, if not repaired, has the potential to generate mutations in somatic
or germline cells, which ultimately can alter cellular phenotype, cause dysfunction
and/or disease. To prevent such deleterious effects and maintain genome stability,
cells have evolved a number of mechanisms to detect and repair the various types of
damage that can occur to DNA. no matter whether this damage is caused by errors
in. replication or by the environment (Tabic 3.6). Well-known mechanisms include
base-excision repair, nucleotide excision repair, mismatch repair, homologous
recombination, and non-homotogous end-joining (Pig. 3.21). Additionally, the cells
possess mechanisms in which damage is directly reversed most often by a single repair
protein without the incision of DNA backbone.
Table 3.6. Types of DNA damage and the mechanism of ils repair

Methylated (0s or N1') Guanine Direct repair

Oxidized/Deaminated bases Base excision repair
Bulky DNA lesions Nucleotide excision repair
Mismatched bases Mismatch repair
Double-stranded DNA breaks Non-homotogous end-joining
Double-stranded DNA breaks Homologous recombination

DNA Polymerase Proofreading

DNA polymerase corrects nucleotide synthesis errors prior to extension through
proofreading mechanism. The polymerase checks whether the newly added base has
3.3. DNA Damage & Repair 123

paired correctly with the base in. the template strand. If an incorrect base has been
added, die enzyme removes and replaces it (Fig- 3.14 A). This is performed by the 3’
exonuclease activity of DNA pol. Once the incorrect nucleotide has been removed, it
can be replaced by the correct one.
A DNA proofreading

...and repteces ii wifri

The DNA potymerase
an r’con Ec: base me? ihe correct base befc-re
be added w the growing mmediaiEty excises jxoceecfing wiiti
The incofreci base..
chain. repl ica bon

B Nfomalcn repair

ThE rnisrnaiEfi repair

A DNA priymerase In ire las? srep-,
proteins me misrnaiched

f
adds ihe commi DNA ligase repairs
base and some adjacent bases. ! Lhe remaining nick
bases.

Fig. 3.14. DNA repair mechanisms..(A) proofreading activity of DNA pol. During replication, me DNA
polymerase enecks for incorrect oases in the new DNA strand and immediately replaces them witn
correct ones. (B) DNA mismaten repair. After replication, mismaten repair proteins searenfor incorrect
oases tn at were missed t?y DNA pol and replaces mem

Exonuclease activity improves the accuracy of DNA pol. The combination

of exonuclease proofreading and polymerase activities of DNA pol make the copying
of DNA very accurate.

DNA Repair by Excision

Excision repair is the most important DNA repair mechanisms in eukaryotic cells.
It is involved in repairing of a wide variety of chemical allo rations to DNA. Excision
is the general mechanism by which repairs are made when one of the double helix
strands is damaged. The damage on one of the strands is removed. The resulting, gap
is then filled in by the synthesis of a new DNA strand using the intact strand as a
template.. There arc three types of excision repair:
1) mismatch repair;
2) base-excision repair:
3) nucleotide excision repair.
DNA mismatch repair (MMR) corrects DNA. mismatches generated during DNA
replication, thereby preventing mutations from becoming permanent in dividing
cells. MMR is a highly conserved pathway that plays a. pivotal role in maintaining
genomic stability. The specificity of MMR is primarily for base-base mismatches
124 Chapter 3. Sene expression and protein synthesis

and insertion and deletion mispairs generated during the replication. During
MMR. enzymes delect distortions caused by mismatched bases and subsequently
remove the incorrectly paired nucleotide and replace it with the correct nucleotide
(Fig 3.I.4 B).
Base-Excision Repair (BER) involves the recognition and removal of a single
damaged base. HER corrects DNA damage from oxidation, deamination, and
alkylation. These lesions cause little distortion to the DNA helix structure. HER
mechanism requires a family of enzymes called glycosylascs. The goal of DNA
glycosylascs is to locale quickly and efficiently the aberrant base amongst a huge
excess of the normal ones. Glycosylases cleave the bond between deoxyribose and a
modified or mismatched DNA base. At least II distinct mammalian DNA glycosylascs
arc known, each recognizing a few related lesions, frequently with some overlap in
specificities. DNA glycosylasc recognizes and removes the damaged base forming an
abasic site (AP site) which is repaired by AP endonuclease before the nucleotide gap
in the DNA. strand is filled by DNA pol (Fig. 3.15).
Nucleotide Excision Repair (NER) is a widespread mechanism used by mammals to
remove bulky DNA lesions such as those formed by UV light {Fig. 3.16), environmental
mutagens, etc. The modified nucleotides cause a significant distortion in the DNA
helix structure.
In NER, the damaged bases arc recognized and subsequently cleaved out within
a string of nucleotides by endonucleases and replaced with DNA as directed by the
undamaged template strand by DNA pol (Fig. 3.17).
Direct Reversal Repair is one of the lesser-known mechanisms of repair where
the damaged lesion is repaired directly by specialized proteins. In contrast to
excision repair mechanisms discussed above, direct reversal of DNA damage, docs
not involve the process of breaking Lhc phosphodicstcr backbone of the DNA and,
thus, docs not require a template. One of the examples of the reversible DNA
damage is the damage caused by alkylating agents reacting with DNA. Alkylating
agents arc carcinogens that generate a broad spectrum of DNA lesions. DNA
alkylation damage can arise from exogenous or endogenous sources. Elimination of
the damage by direct reversible repair is achieved by two major types of proteins: Or'
methyl guan inc methyl transferase (MGMT) and a Ip ha-kcto-glutaratc-depend cm
and iron-dependent oxygenases.
Methylation of guanine O6 position confers the greatest mutagenic and
carcinogenic potential. O^methylguanine changes guanine's hydrogen-bonding
pattern and can confuse the cell into pairing it with T. During replication, one of (he
strands ends up with A-T, where a G-C should have gone.
The suicide enzyme methylguamne methyltransferase (MGMT/MTasc) can
remove the abnormal methyl group by transferring it to the sulfhydryl group of a
cysteine in the active site (Fig. 3.18). Importantly, unlike most enzymes, this enzyme
is deactivated by the reaction, it can no longer participate in transferring methyl
groups.
3.3. DNA Damage & Repair 125

Damaged tease —

■ ■ > I lx I iIi i 3
. JJLSJLUL5JJUJL 6.
DMA glycosylase

APsite^-... "

i I ■ 3
3Ai±iAiiiiii5
AP endonuclease

5TST b d’6 iff 3'

DMA polymeiase

1 DMA ligase

Fig. 11 5. Schematic illustration ot ON A repair by base excision. BER is initiated by a DNA glycosylase
specifically recognizing and binding a base lesion, upon encountering a substrate base the
glycosylase flips me base out and generates an AP site, whicn is recognized by an AP endonuclease
tnat hydrolyzes the pnospnodiester bond 5r to the lesion. Polymerase fl fills in me nucleotide gap,
which is subsequently seated by the DNA ligase, restoring the original base sequence

O O 0 0

Fig. 116. Thymine dimer formation under UV

126 Chapter 3. Sene expression and protein synthesis

Damaged nudsotide ■—

d n ■■ o
?9aaPD9?DD9

’ ^...-------- Endonucleases

□ c □ □ □ □ □ aMl 3
■ millin’

51

...................................................................................
3'
DNA polymerase I

5'
1 DNA ligase

3’

3' 5’

Fig. 3.17. The schematic illustration of nucleotide excision DNA repair HER proteins recognize the
damaged DNA, excise the damaged nucleotides, and repair the DNA strand Dy re-syntnesis/iigaiion to
restore the integrity of the DNA molecule
3.3. DNA Damage & Repair 137

Active MTas* Inactive WTase

H—N H-—N
Q6MeG 'i G
H H

Fig. 3.18. Direct revere a! repair. OG mefflyl guanine (OGMeG) is mutagenic because polymerases
frequently misinsert T opposite 0EMeG in stead of C, therefore the metnytaiion alters hydrogen-
bonding patterns of guanine in duplex DMA. MGMT restores tne original guanine Dy transferring the
methyl from 0GMeG to its active site

Other DNA Repair Mechanisms

DNA double-stranded breaks (DSB) are am one the most dangerous forms of
DNA damage. DSB occurs when both strands of the double helix are broken, leaving
no intact template strand for repair. Unrepaired' DSBs can result in apoptosis or
chromosomal aberrations including translocations and deletions, which can result in
a loss of heterozygosity. These chromosomal aberrations arc associated with genomic
instability and can result in carcinogenesis. DSBs arc repaired by non-homo logo us
end joining. In this case, DNA ligase joins the damaged ends. DSBs and single-strand
breaks and gaps can be repaired by homologous recombination, in which sections from
the damaged DNA arc exchanged with an undamaged duplex. Lesions that cannot be
repaired by other methods, such as those spanning both strands of the duplex (formed
with cross-linking agents), are also repaired by homologous recombination.
Although genetic variation is important for evolution, the su rvival of the individual
demands genetic stability. DNA repair is essential for every organism, and when there
arc deficiencies in DNA repair, mutations become a whole lol more common that can
eventually rapidly change DNA sequences.
128 Chapter 3. Sene expression and protein synthesis

Fig. 3 J 9. General scheme of DNA repair systems

Hereditary Diseases
Many genetic disorders arc caused by genetic defects of DNA repair mechanisms
Jk/w/cnwa p^wew/ftswm (XP) is the classical human disorder caused by defective
nucleotide excision repair of DNA damage, including pyrimidine dimers induced by
UV. Signsand symptoms of the disease may include: severe sunburn when exposed to
only small amounts of sunlight, irritation, rough-surfaced growths (solar keratoses),
and skin cancers. In the most severely affected cases, there is progressive neuronal
degeneration as well. Treatment is supportive. Radiation is contraindicated.
Other hereditary diseases characterized by genetic defects of DNA repair
mechanisms include Nijmegen breakage syndrome, Werner syndrome, I?loom
Syndrome, Fanconi anemia, Cockayne syndrome, trichothiodysirophy. These rare
3.4. TranscriptiDAz RNA Synthesis 129

disorders share many clinical features such as growth retardation, neurological

disorders, premature aging, skin alterations including abnormal pigmentation,
pathological wound healing as well as an increased risk of developing different types
of cancer.

3.4. TRANSCRIPTION: RNA SYNTHESIS

A gene is a. section of DNA that codes for a particular protein and determines a
certain trait. A gene is the basic unit of heredity.

DNA Dependent RNA Polymerase

The central dogma of molecular biology is DNA codes for RNA. which codes for
proteins i Fig. 3.20). The process of synthesizing RNA from the genetic information
encoded by DNA is called transcription. DNA dependent RNA polymerase or RNA
pol is an enzyme that is responsible lor copying a DNA sequence into an RNA
sequence during the transcription. Cellular RNA polymerases are multisubunit
enzymes. Eukaryotes have four different RNA pols. Each polymerase is responsible
for the synthesis of a different class of RNA. Three (I, II, and 111) are required for
transcription, of nuclear genes and the fourth is for transcription, of mitochondrial
genes. RNA pol I transcribes ribosomal RNA (rRNA), pol 11 transcribes mRNAand
pol III tRNAand several small RNA’s.

Fig. 320. The simplified scheme of me central dogma

The RNA is synthesized from a single strand or temp late/sense strand of a DNA
molecule. The opposite DNA strand is called a coding strand, because its nucleotide
sequence corresponds to the sequence of the RNA transcript produced. The section
of DNA that is transcribed into an RNA molecule is called a transcription unit
A transcription unit codes the sequence that is translated into protein.
130 Chapter 3. Gene expression ano protein synthesis

Transcription occurs in three sequential stages: initiation, elongation, and

termination.

Stages of Transcription: Initiation

Transcription begins with the RNA polymerase and accessory proteins, TFs,
binding to DNA in the promoter region, which is in the immediate vicinity of
the transcription start site. Transcription factors (TFs) arc the proteins that help
determine which DNA sequences should be transcribed and precisely when the
transcription process should occur. The structure of eukaryotic promoters is generally
complex, and they have several different sequence motifs, which include the TATA-
box, initiator (Inr-box), CAAT-box and GC-box (Fig. 3.21). TATA-box is usually
located 25-35 base pairs upstream of the transcription start site, the CAAT-box and
the GC-box arc located from 40 to 200 base pairs upstream of the transcription start
site. Each of these elements serves as a binding site for specific TF. TFs recruit RNA
pol and control and regulate the transcription of DNA into mRNA. The RNA pol
Il is associated with six general transcription factors, designated as TF11 A, TFIIB,
TFI1D, TFI1E, TFIIF. and TFIIH (Table 3.7). In addition to the promoter, other
ejj-acting DNA sequences regulate RNA polymerase II transcription, e.g., enhancers
and silencers (discussed below).

region ileadefi region (trailer^

fig. 3.21. Eukaryotic core promoter elements. Some upstream regulatory elements that can participate
in transcription by RNA polymerase h are depicted rnr and a coding region with introns and exons and
poiy(A) tail. The first base on the DMA where transcription actually starts is iabeied +1. Sequences that
are upstream of me first base of me transcript are marked with negative numbers. Sequences that are
downstream of me first base of the transcript are marked with positive numbers

Table 3.7. Summary of eukaryotic RNA polymerase II general transcription factors

TF
TRIA 3 Stabilizes binding of TATA-binding protein (TBPI and TFIIB
TRIB 1 Binds TBP.. selects start site, recruits RNA pol II
TRID 12 Interacts with regulatory factors
TBP 1 Subunit of TFIID. specifically recognizes the TATA box
TRIE 2 Recruits TFIIH
TRIF 2 Binds RNA pol II and TRIB
TRIH 9 Unwinds promoter DNA, phosphorylation of RNA pal II
RNA poll II 12 Catalyzes RNA synthesis

When the complex, of TFs and RNA pol is assembled (Fig. 3.22 A), DNA is
unwound to expose the template strand for the synthesis of an RNA molecule.
3.4. Transcription: RNA Synthesis 131

Transcription
RNA polymerase ll start site

strand

5’ I I I I I
A A - I Q

’GA G-iA
31 l i ill ill i i

promoter Template strand

□NA

Fig. 122. Transcription: RNA syiitnesis. (A) eukaryotic transcription initiation. Simplified model of
protein complex assembly on DNA is shown; (B) RNA synthesis. The RNA poi moves stepwise along
the DNA, unwinding me DNA helix at its active site, it adds nucleotides one Dy one to me RNA chain
al me polymerization site using an exposed DNA strand as a template. The incoming nucleotides are
in me form of ribonucleoside triphosphates (ATP, uTP, CTP, and GTF'i. and the energy stored in their
phosphate-phosphate bonds provides the driving force for me polymerization reaction

Stages of Transcription: Elongation

On.cc the polymerase 11 has begun elongating the RNA transcript, most of the
general transcription factors arc released from the DNA so that they are available
to initiate another round of transcription with a new RNA polymerase molecule.
Typically, many RNA polymerases simultaneously transcribe the same gene. This
means that many RNA. copies of the same gone can be made in a short time.
R NA pol links ribonucleotides together in a 5’ to 31 direction, i.c.. adds nucleotides
to the 3' end of the growing chain (Fig. 3.22!?). As in DNA replication, the nucleotide
sequence ofthe RNA chain is determined by the complementary base-pairing between
incoming nucleotides and the DNA template. The growing RNA chain is extended
by one nucleotide al a time. The substrates arc nucleoside triphosphates (ATP. CTP,
132 Chapter 3. Sene expression and protein synthesis

UTP, and GTP). As in DNA replication, a hydrolyzis of high-energy bonds provides

the energy needed to drive die reaction forward.
Nucleotide addition continues until specific termination signals arc encountered.

mRNA Capping
Capping of the prc-mRNA is the addition of 7-mcthylgtianosinc (m7G) to the
S’-end (Fig. 3.23).

323. Cap structure, wnile ine pre-mRNA is stilt feeing synthesized, a 7-methytguanosine cap is
added to the 5-end of we growing transcript by capping enzyme

Capping is the first modification made to RNA.and lakes place co-transc optionally
in the nucleus when the first 25-30 nucleotides arc incorporated into the nascent
3.4. Transcription: RNA Synthesis 133

transcript. Three enzymes, RNA triphosphatase, guanvly] transferase, and

methyl transferase arc involved in the addition of the cap to the mRNA. The 5' m7G
cap is an evolulionariiy conserved modification of eukaryotic mR NA. In addition to
its protective function of the 5’-end from exonuclease cleavage, it has an essentia! role
in cap-dependent initiation of protein synthesis and pre-m RNA splicing.

Stages of Transcription: Termination and Polyadenylation

Transcriptional termination of eukaryotic mRNA genes occurs when RNA pol 11
encounters specific termination signals. The canonical polyadenylation signal (PAS)
3’-AAUAAA-5’ (or less frequently AUUAAA) directs the incorporation of the
termination and polyadenylal ion signal (Fig. 3.24). The signal is located upstream of
the cleavage site and is recognized by the multi meric cleavage and polyadenylation
specificity factors. The moment a poly(A) signal is transcribed by pol IL cleavage
factors bind to these elements in the nascent pre-mRNA. Another canonical sequence
dement, GU-rich sequence (or downstream sequence element, DSE), is located
downstream of the cleavage site, and promotes the efficiency of 3’-end processing.
Importantly, polyadenylalion is a process of template-independent addition of a
polv(A) tail to the .V-end of mRNA by poly(A) polymerase (PAP). In mammalian
cells poly (A) tail is long and consists of more than 200 nucleotides. They arc degraded
in the cytoplasm by continuous shortening throughout the lifetime of the mRNA.

General consensus canonical polyfAi sequence

( 0-20 nt l 15-30 nl . 0-20 nt

-------- jta LI Rich AAUAAA CA fctl/ll ririi - *—
USE Poly(A) signal Cleavage site DS~

Fig. 324. Canonical consensus poiy(A) sequence. The canonical consensus has elements sucn as the
AAUAAA polyadenytation signal, cleavage site -15-30 nucleotide downstream of PAS, G/GU rich DSE
and a. U-ricti USE

In general poly(A) tails play crucial roles in maintaining mRNA stability and
turnover, transport of mRNA from nucleus to cytoplasm, and translation efficiency
of mRNA. Nearly all eukaryotic mRNAs have a poly (A) tail al their 3’-end, with the
exception of histone mRNAs, which lack a poly(A) tail.
In humans, pre-mRNAs can be polyadcnylatcd in several different ways due
to the existence of more than one polyadenylalion site, allowing a single gene to
encode multiple mRNA transcripts. More than half of the genes in the human
genome arc alternatively polyadenylated. Alternative polyadenylalion plays an
important role in cell growth, proliferation, and differentiation. Importantly,
alternative polyadenylation potentially can alter the dynamicsand properties of an
RNA transcript affecting stability, translation and/or subccllular localization. If
for example, the alternative site lies upstream of the usual stop codon, a truncated
RNA transcript that tacks one or more exons is produced. This eventually leads to the
alteration in protein sequence and function. Abnormal alternative polyadenylation is
134 Chapter 3. Sene expression and protein synthesis

associated with a wide range of human diseases including thalassemia, Huntington’s

disease, etc. (Table 3.8). Therefore, regulation of alternative poly adenylation is
important in gene expression.
Table 3.8. The summary of alternative poiyadenylation related diseases

Endocrine Steroid agenesis STAR Br-cAMP stimulates distal PAS usage

Diabetic nephropathy HGRG-14 H igh-g lucose level leads to distal PAS

usage

Type 2 drabetes TCF7L2 Increased different isoforms by usage of

intra nic PAS

Hematological p-Thalassemia HBB Elongated 3UTR region and

transcription termination defects by
mutation son polyadenytation site

a-Bialassemia HBA1

Tumorigenic Proliferative conditions RBX1 Hyper-activated mTOR leads lo usage of

proximal PAS

Colorectal cancer DMKN. PDXK, 3'UTR shortening has occurred during

and PPIE tumorigenesis

Glioblastoma CCND1 Knockdown of CPSF5 induces 3TJTR

shortening

Infection and B-ce-ll differentiation IGHM CSTF2 leads to proximal PAS usage
immunological

T-cell activation NF-ATC1 Upregulation of CSTF2 stimulates 3'UTR

shortening during T-cells activation

Systemic lupus GlMAPS Disruption of proximal PAS by mutation

erythematosus

IPEX syndrome F0XP3 Mutations on first PAS

Neurological Parkinson's disease SNCA PD risk factor induces shorter isoform

Oculopharyngeal CCND1 PABPN1 suppresses usage of proximal

muscular dystrophy weaker PAS

Huntington's disease HTT Depletion ol CNOT6 induces isoiorm

shift

STAR. sWraidogBiiic acuta ragulalory: Br-cAMP, bromoadBiiosina 3'.5'-cyclic moflopkc&phatB; PAS. poly-adenylarTofi signal;
HGRGrl4_nigh-gluCDSB-regulala<l gane 14: TCF7L2, transcription factor 7-lika 2: HBB, nemoglsDin p: UTR. untrarcslaiBd r&giort;
HBA1. hemoglobin a 1 RBX1. ring-box 1: DMKN. dDrmakina; PDXK. pyridoxal kinasB: PF'lE, psplidyipiopyl isomsrasB E;
GGNDl, cyclin 01: CPSF5. cfeavags and potyadenylalion spfldf icily factor 5; IGHM, immunogloiRjlin iwavy constant mu;
GSTF2. ctoavags stimulation factor 2: NF-ATC1. nuclear factor cl adivalC’d T-cells 1; GlMAPS, GTPasa. IMAP family member
5; lPEX_ immuiw dysfunctions. polyendocrinopalliy. wiferopaBiy, X-linked: FOXP3. lorknead box P3: SNCA. synuclein ll PD,
Parkinson's disease; PABPN1. pcJy A-binding protein nuclear 1; HTT. Huntington's tfisBaso; CNOTG. CCR4-NOT transcription
complex subunit 6; UTR, unti aiisiatBd region.
3.4. Transcription: RNA Synthesis 135

The actual termination of transcription by RN A pol 11 occurs up to several hundred

bases downstream of the 3’-end processing site even though both processes arc
intimately linked. RNA polymerase disengages from the template DNA and releases
its transcript. Importantly, mutaiioiis of 3’ end processing factors may result in
transcription termination defects that can be associated with various human diseases,
such as thrombophilia, thalassemia and cancer.

Transcription: Removal of Introns

in eukaryotes, when RNA is transcribed from DNA, it often contains additional
noncoding sequences (intron) within the coding sequence (exon). Exons correspond
to protein-coding sequences (ex-on signifies that they arc expressed), and introns
correspond io noncoding regions. This primary RNA molecule transcript is referred
to as precursor mRNA (pre-mRNA). Before it is transported into the cytoplasm to
serve as a template for protein synthesis, the pre-mRNA undergoes processing within
the nucleus. The introns are removed to produce the mature mRNA molecule by a
process known as RNA splicing (Fig. 3.25 A). The splicing reaction occurs in several
steps and is catalyzed by the spliccosomc, a targe ribonucleoprotein (RNP) complex
that forms from the ordered assembly of small nuclear ribonucleoprotein (snRNP)
particles onto each, intron. Regulatory elements located within introns and exons guide
the splicing complex, the spliccosomc, and auxiliary RNA binding proteins to the
correct sites for intron removal and subsequent exon joining. Misregulation of splicing
causes a number of diseases, including Familial Dysatnonomia, Neurofibromatosis
type I, Frasier syndrome, and atypical cystic fibrosis.
For most eukaryotic genes, pre-mRNA can. be spliced in a number of different
ways (alternative splicing}, thereby creating different combinations of exo ns in the final
mature mRNAs (Fig. 3.25 B) (discussed in Ch. 3.6}. Thus, alternative splicing allows
production of a large variety of proteins from one gene.
Once these steps have been successfully completed, a mature m RNA can travel out
of the nucleus and be used to make a protein.
3'-end processing is intricately coupled to transcription and splicing, and also
regulates the type, and the amount of mRNA and protein levels of a particular gene.
Thus, mRNA 3’-cnd formation links transcription of a gene with the translation of its
mRNA.
136 Chapter 3. Sene expression and protein synthesis

pre-m R NA transcript
A
2 miron Exon 3

I
Excm 1 I .__ E»0fi2 Exon 3 ~

Spliceosome

/ X

[ Exon 1 | [ Exon 2 ] [ Exon 3 ] ] Exon 4 |

Alternative splicing

Exon I
/
| Exon 2 j Exon 3 ] |
\
Exon 1 | Exon 2 | Exon 4~

Translation

Protein A Prolein B

Fig. 325. RNA spicing. The spliceosome is assembled from small nuclear ribo nucleoproteins
{snRNPs}. snRNPs recognize conserved sequences in splice sites, remove the introns, and past the
exons togetner. (A) Sc nematic diagram of the steps of spliceosome assembly and intron removal,
introns are removed by RNA processing in wnicri the intron is looped out and cut away from the exons
by snRNPs, and tne exons are spliced togetner to produce tne translatable mRNA (B}. When different
coding regions of mRNA are spliced out different variations of matured mRNAs that encode different
proteins will eventually occur
3.4. Transcription: RNA Synthesis 137

Questions and situational problems

1. Which of the Mowing arc not the components of RNA?
1) thymine;
2) adenine;
3) guanine;
4) cytosine.
2. In a DNA molecule:
1) adenine and thymine arc equal to the amount of guanine and cytosine;
2) adenine and guanine arc equal to the amount of thymine and cytosine;
3) adenine and uracil are equal to the amount of guanine and cytosine;
4) adenine and guanine arc equal to lhe amount of uracil and cytosine.
3. Amrfcrma i&men/osiijn patients have a 10,000-fold increase in the risk of developing
skin cancer. These patients have to avoid exposure lo UV light. Please explain why; To
answer the question please:
1) name lhe process that is affected and provide a scheme;
2) indicate template, substrates, sources of energy that are involved in lhe process:
3) propose the consequences of the process.
4. Why do introns need to be cut out from an RNA transcript? To answer lhe question
please:
1) explain what introns arc:
2) draw a scheme of intron removal.
5. A select mutation is causing a cell lineage to be unable to replicate DNA successfully.
When observed under a microscope, researchers observe that the DNA is able to be
separated, but the template strands keep coming back together before lhe new strands
can be replicated. Based on this observation, which protein involved in DNA replication
is most likely mutated? To answer the question please:
1) draw a scheme of DNA replication:
2) name the proteins that arc required for DNA replication:
3) propose the consequences of the process.
6. What is the main reason for there being both a leading and a lagging strand during DNA
replication? To answer the question please:
1) draw a scheme of DNA replication and mark the direction of replication fork
movement:
2) name the proteins that arc required for DNA replication.
7. What enzyme synthesizes small primers for DNA polymerase to bind to so it can initiate
DNA replication. What are these primers made of? To answer the question please:
1) name the proteins that arc required for DNA replication;
2) draw a scheme of DNA replication;
3) design a primer for this DNA fragment; 5’-AATTGGCA-3\
8. Consider the following DNA sequence: 5’-AAATGCCAC-3\ Identify the correct mRNA
transcript:
1) 5 -TTTACGGT-3’;
2) 5*-UUACGGG-3’;
315-UUUACGGG-31;
4} S^-UUlJACGGUG-l’.
9. What is the role of RN A poly merase? To answer lhe question please:
I > name three types of RNA in a cell and their functions;
2) draw a scheme of RNA synthesis;
3) name the enzymes required for RNA synthesis.
138 Chapter 3. Sene expression and protein synthesis

Primer 5’-CUU-3’ is being used to replicate this piece of DNA. What strand this
primer will anneal to: the upper or the lower? Write what product of RNA replication
would be the first live nucleotides that had been added onto the primer by DNA pol.
Mark the 5’ and 31 ends.
1 L lien? is a short stretch of replicating DNA. The lop strand is the template. Assume
the are the RNA primers. Numbers *1,2, 3, 4* indicate parts of four Okazaki
fragments.

Which was made first, the Left or the Right fragment? Which primer will be the first
to be removed. Left, Middle, or Right?
When the primer in the middle is removed and filled in with DNA, the fragments
must then, be joined. Which enzyme joins the fragments? Where is the final,
connection made: a or b?
12. A. diagram of DNA replication is shown below; Redraw the diagram into copybook
and fill in the empty boxes in the figure.

5' 3'
3.4. Transcription: RNA Synthesis 139

13. A scheme of DMA replication is shown below. Rewrite the diagram into copybook and
fill in I he empty boxes in the figure.

The leading strand is

synthesized continuously
in ire 5- 3’drection by
DNA polymerase.

Replication fork
RNA primer

Primase Okazaki fragment

being matte
5 ONA----------
3t polymerase
Parental DMA

After tte RNA primer is

replaced by DNA (by anzeher
DNA polymerase, rot EfjOWiL
DNA ligase joins the Okazaki DMA ligase
fragmertf Id the growing strand.

Overall direction of replication

14. A scheme of UNA replication is shown below. Redraw the diagram into copybook and
fill in I he empty boxes in the figure.

A A
The leading strand is
Single-strand binding
Helicases unwind the synthesized continuously
proteins stabilize the
parental double helix. in the 5 W direction by
unwound parental DNA
DNA polymerase.

i i i ii •1
The lagging strand is
synthesized discontinuously.
Okazaki fragment Pnmase synthesizes a short
RNA primer, which is
extended by DNA polymerase
to form an Okazaki fragment

After the RNA primer is

replaced by DNA ■;tv another
DNA pdymerase, hot shown.!,
DNA ligase joins the Okazaki
fragment to the growing strand.

Overall direction of replication

140 Chapter 3. Sene expression and protein synthesis

15. A scheme of DNA replication is shown below. Indicate lhe sequence of events that
occur during DNA replication by placing number in the emptied semicircle.

Single-strand binding The leading strand is

Helicases unwind the synthesized continuously
proteins stabilize the
parental double helix. in the 5'“3'diiEction by
unwound p-arental DNA.
DNA poiymerose.

DNA potymerase

Replication fork
The lagging strand is
RNA primer
synthesized discontinuously.
Primase Okazaki fragment Primase synthesizes a short
being made RNA primer, which is
extended by DNA polymerase
5 DNA------------ Lo Form an Okazaki Fragment
3'
potymerase
Parental DNA

After the RNA primer is

replaced by DNA i.by a nether
DNA polymerase, hot shown],
DNA ligase joins the Okazaki DNA ligase
fragment to the growing strand.

Overall di rected of replication

16. A diagram of splicing is shown below. Rewrite the diagram into copybook and fill in the
boxes with the question mark in the figure.
pro-mRNA transcript

l
ST
1
m
> Exon 2 Exon 3
3.5. Genelic Code Properties 141

17. A scheme of transcription is shown below E Rewrite the diagram into copybook and fill in
the boxes with the question mark in the figure. Mark the 5'- and 3’-ends.

Nontempiate strand

Direction
□f synthesis —
■ ■ . i rrTTliQ
#111

s a a G
promoter J-l t-L Template strand
□ RNA polymerase

3.5. GENETIC CODE PROPERTIES

Genetic code is the sequence of nucleotides in DNA and RNA that de [ermines the
amino acid sequence of proteins.
Genes that provide instructions for proteins arc expressed in a two-step process
(Ch. 3.4).
► In transcription, the DNA sequence of a gene is <rewritlen* in RNA.
► In translation, the sequence of nucleotides in the mRNA is ^translated* into a
sequence of amino acids in a polypeptide (protein chain).
Some of the most important properties of genetic codes are:
► the code is a triplet codon. Three adjacent nucleotides constitute a unit known
as the codon, which codes for an amino acid (Fig. 3.26?. The nucleotides of
mRNA are arranged as a linear sequence of codons. Usually, the AUG codon,
in addition to coding for methionine, is found al the beginning of mRNA and
indicates the start of a protein. In the genetic code , a stop codon (or termination
codon) is a nucleotide triplet within mRNA that indicates the end of a protein,
i.c., a termination of translation. There arc three different stop codons in RNA:
UAG (camber*), UAA Gochrc*) and UGA(«opal*);
► the genetic code is non-overlapping. The genetic code is read sequentially, i.c.,
one codon at a time. Codons do not share their bases: therefore, the code is non­
overlapping;
► the genetic code is nearly universal. The genetic code is pretty similar in most of
the organisms;
► the genetic code is continuous and conimaless. There is no signal to indicate the
end of one codon and the beginning of the next, i.c., no nucleotide or comma (or
punctuation) is present in between two codons;
► the genetic code is unambiguous. Each codon specifics one amino acid only;
► the genetic code is degenerate. One amino acid may be coded by more than
one codon, i.c., the same amino acid is coded by more than one base triplet.
For example, the three amino acids arginine, alanine and leucine each have six
synonymous codons (Fig. 3.26);
142 Chapter 3. Sene expression and protein synthesis

Second fetter
u C A G

uuu Pte
ucu UAU
Tyr
UGU
Cys
u
u uuc ucc Ser
UAC UGC c
UUA UCA UAA Stop UGA Stop A
Leu
UUG UCG UAG Stop UGG Trp G

cuu ecu CAU CGU U

His
c
cue ccc Pro
CAC CGC C
Leu Arg
CUA CCA CAA CGA A
Gin
CUG CCG CAG CGG G

AUU ACU AAU AGU U

Asn Ser
M.
AUG lie ACC
Thr
AAC AGC c
AUA AGA AAA AGA A
Lys Arg
AUG Met ACG AAG AGG G

GUU GCU GAU GGU U

ASp
GUC GCC GAC GGC c
G Vai Ala Gfy
GUA GCA GAA GGA A
Glu
GUG GCG GAG GGG G

Ry. 326. Tne genetic code table

► the genetic code has polarity. The code is always read in the 5’ -* 3’ direction:
► the- gene and the polypeptide it codes for arc collinear. The sequence of nucleotide
pairs in a gene specifics a coll incar sequence ofamino acids in its polypeptide product.

Ammoacyl-tRNA Synthesis
Amino acids arc the substrate for protein synthesis, but they cannot be used
directly. They must first bind iRNAinordcrtobe used in protein synthesis (Fig. 3.27).
lRNA is an adapter molecule typically 70 to 90 nucleotides in length (discussed in
Ch. 3.1 ). tRNA contains rare bases in addition to the common bases. The rare bases
are nucleotides, which arc unusual either by the nature of the base, which is often a
methvfated base, or even by the nature of the bond between the base and the sugar.
These modi float ions arc catalyzed by various specific enzymes after the synthesis of
tRNA. The 3’ terminus of all tRNA has a conserved CCA-3’ sequence to which amino
acids bind. The main funcLion of tRNA is the transfer of ami no acids during protein
synthesis.
3.5. Genelic Code Properties 143

n
n

------ 'GO Qi——

mRNA
51 Codon

Fig. 327. Alanine tRNA or tRNA3’3 (codon GCC). Anticodon (CGG) is indicated Dy me red circle. When
a FINA recognizes and binds to its corresponding codon in the ribosome, the tRNA transfers tne amino
acid to tne end of the growing amino acid chain

The function of aminoacyl-tRNA (aa-tRNA or charged tRNA) synthesis is to

precisely match amino acids wit htRNAs containing the corresponding anticodon. This
is primarily achieved by the direct attachment of an amino acid to the corresponding
tRNA by an aniinoacyl-tRNA synthetase (Fig. 3.28). The amino acid first reacts with
ATP to form aminoacyl-AMP and pyrophosphate. The ami noacyl-AMP binds to
tRNA to produce the charged tRNA plus AMP. The summation of these steps is:

Amino acid+tRN A+ATP-Aminoacyl-tRNA+AM P+PP..

1M Chapter 3. Sene expression and protein synthesis

o o o o

I
c™o (Aminoacyl-tRNAj
I
HO— R
I
NH/ Aminoacyl-tRNA synthase

Rg. 3.28. Binding of an amino acin to its specific tRNA. Tne aminoacyt group is esterified to the 31
position of tne terminal adenylate residue

Twenty different aminoacyl-tRNA synthetases exist, one for each amino acid.
Therefore, each iRNA is specific of amino acid, i.c., it can. bind (or * accept M only that
particular amino acid. Thus, tRNA-dj denotes an RNA specific of alanine, capable of
binding and transferring alanine, iRNA^ denotes an RNA specific of phenylalanine,
etc.

3.6. TRANSLATION: PROTEIN SYNTHESIS

All mRNAs arc read in the 5’--*3’ direction. Polypeptide chain is synthesized from
the amino to the carboxj I terminus. Each, amino acid is specified by a codon in the
niRNA, according to a nearly universal genetic code (discussed above). The mechanism
of protein synthesis is the same in all cells. Translation is carried out on ribosomes,
with [RNAs serving as adaptors between the mRNA template and the amino acids
being incorporated into protein. Additionally, various proteins arc involved in protein
synthesis (Table 3.9).
3.6. Translation: Prolan Synthesis 145

Table 3 9 The factors involved in eukaryotic translation

Proteins <ief3,cfU, eF<G
Elongation Proteins: eEF-1, eEF-2

Termination Proteins:eRF1 and eRF3

Translation proceeds in three phases:

► initiation (or ^beginning*): in this stage, the ribosome gets together with the
mRNA and the first lRNA so translation can begin;
► elongation: in this stage, amino acids arc brought to the ribosome by tRNAs and
linked together to form a chain:
► termination: in this stage, a stop codon is reached, the ribosome releases the
polypeptide.

Ribosome
Ribosomes arc organelles present in both prokaryotic and eukaryotic cells. The
main function of the ribosome is to synthesize proteins. Ribosomes can be found
floating within the cytoplasm or attached to the endoplasmic reticulum (ER).
Importantly, the location of the ribosomes in a cell determines what kind of protein
will be produced. The ribosomes that are floating freely throughout the cell, synthesize
proteins that reside in the cytosol. The bound ribosomes synthesize proteins of the
endomembrane system as well as proteins secreted from the cell. The ribosomes arc
made up of small and large subunits. In ribosomes, approximately two-thirds and
one-third of the mass consist of RNA and protein, respectively (discussed in Ch. 3d).
The ribosomal structure is highly conserved between all species. Interestingly, a
mammalian cell may contain as many as IO million ribosomes, but each ribosome has
only a temporary existence. Ribosomes can link up amino acids at a rate of 200 per
minute.
The ribosome has three main sites for tRNAs: the A-, P-, and E-sites (Tig. 3.29,
left panel). They span the space between the small and large subunits. The mRNA
is bound to the small ribosomal subunit. The large subunit contains the pc pt idyl
transferase site where the acceptor ends of two tRNAs come together to transfer the
nascent peptide to the incoming amino acid (Fig. 3.29, right panel).
Exit lumel

A site ■ Aminoacy! tRNA

tending site)

Large subutel
mRNA
tending
Small s_tunl

Fig. 329 Schematic models showing me binding sites ol me ribosome. Each ribosome has a binning
site for mRNA and three binding sites for tRNA molecules. The P site holds me lRNA carrying the
growing polypeptide chain. The A site carries the tRNA with the next amino acid. Discharged tRNAs
leave the ribosome at the E site. 28SRNA is me catalyst for peptide bond formation. The two subunits
assemble to translate mRNA and disassemble when translation is complete
146 Chapter 3. Sene expression and protein synthesis

Initiation of Translation
Initiation of translation is a complex process when initiator tRNA, 40S, and 60S
ribosomal subunits arc assembled by eukaryotic initiation factors (elFs) into an 80S
ribosome al the initiation codon of mRNA (Fig. 3.30). Translation docs not simply
begin al the 5’ end of the mRNA; instead it starts at specific initiation sites.1 mRNA
of eukaryotes has 5’-cap structure and 3’-polv(A) tail (discussed in Ch. 3.4), both
of which arc important for the initiation of translation, in eukaryotes, a cap-binding
protein and several other cl Fs assist the movement of the 40S small ribosomal subunit
to the 5’-cap.
Once at the cap, the initiation complex com prising mRNA, the 40S small ribosomal
subunit, cl Fs, Mcl-tRNA.Mcl(initiatormethionyl-tRNA> and nucleoside triphosphates
(GTP and ATP — sources of energy) tracks along the mRNA in the 5’-*3T direction,,
searching for the correct start codon. The AUG codon for methionine functions as
the start codon, in the vast majority of mRNAs. The initiation AUG triplet is usually
separated from the cap by 50-100 nt. The efficiency of recognition of the 5’-proximal
AUG is influenced by a Kozak consensus sequence 5’-GCCGCCRCCAUGG-3’,
where R is a purine (A or G). According to Kozak's rules, the nucleotides around
the AUG indicate whether it is the correct start codon. If the context of the 5’-proximal
AUG is poor, the efficiency of initiation is reduced, and those ribosomes, which fail
to initiate at this site, continue scanning and initiate at the next AUG (leaky scanning)
(Fig. 3.30 A).
Hydrolyzis of GTP provides the energy for the union of the 40S complex with
the 60S subunit to form the 80S initiation complex of eukaryotic cells. Initiation
factors arc then released and translation proceeds by elongation of the polypeptide
chain.

1 The cap-independent initiation is discussed elsewhere.

3.6. Translation: Prolan Synthesis 147

4Cc3

S'-UTR main ORF 3” UTR

5’
AL<3 AUG
I—1

initiation
Efficient of translation
translation

Fig. 130. The model tor initialian of translation in eukaryotes. (A: classical (efficient translation) cap­
dependent initiation mechanism: 40S ribosomal subunits associated with me elFs-GTP-tRNAiMei
complex scan me mRNA for initiation codons, and translation is initiated at the first AUG. Non-
ciassicai (leaky scanning) cap-dependent initiation mecnanism is as follows: the first AUG start codon
can De bypassed and translation can start at tne next AUG codon. (Bi scheme of eukaryotic translation
initiation. Translation begins when an initiator tRNA anticodon recognizes a codon on mRMA The large
ribosomal subunit ioins the small sub unit. The attachment of the aminoacyt-tRNA tome ribosome is an
energy-requiring process. GTP acts as an energy source during translation, at the start of elongation
and during the ribosome's translocation. ORF — open reading frame: uTR-untransiated region
149 Chapter 3. Sene expression and protein synthesis

The AUG Start Codon is Recognized by Methionyl-tRNAWet

Cells contain two different methionine tRNAs (Fig. 331):
► tRNA^ initiates protein synthesis:
► tRNA1**1*: incorporates methionine only into a growing protein chain.

lRNAMet Methionine Met tRNAWat Initiation

tRNAMa! Methionine Met Elongation

Fig. 3.31. tRNA for methionine is of two types. Initiator 1PNA, tRNA.1*4, is used exclusively to initiate
protein chains, and eiongator tRNA, tRNAWcl, transfers mewionine to internal sites in a growing protein
chain

Interestingly, the same aminoacyl-lRNA synthetase charges both tRNAs with

methionine. Only Mel-tRNA.Mca binds at the appropriate site on the small ribosomal
subunit (Psite) to start protein synthesis. The eukaryotic Mct-tRNAj, with, help from
other proteins of the initiation complex, binds directly to the P site. This forms an
initiation complex with a free A site ready to accept the tRNA corresponding to the
first codon after the AUG. Whereas, tRNAMrt binds only to A site on the ribosome.

Elongation of Translation
The translation elongation cycle adds one amino acid at a time to a growing
polypeptide chain according to the sequence of codons found in the mRNA. During
chain elongation, each amino acid is added to the nascent polypeptide chain in a
three-step cycle:
► positioning the correct aminoacyl-tRNA in the A.site of the ribosome;
► formation of the peptide bond:
► translocation of the mRNA by one codon.
As is the case with initiation, a set of proteins, elongation factors (cEFsh arc
required to carry out the process (Table 3.9).
Following translation initiation, an SOS ribosome is poised on. mRNA with the
anticodon of Mei-tRNAj in the P site base-paired with the start codon (Fig. 3.32).
The ribosome is now ready to bind the second aminoacyl-tRNA al the A site, which
will be joined to the initiator methionine by the first peptide bond.
The aminoacyl-tRNA is escorted to the ribosome by an eE F-1, which is complexed
to GTP. When the correct a mi noacyl-tRN A is inserted into the A site of the ribosome,
the GTP is hydrolyzed. The cEF-l bound to GDP is released1.
The second step is the pepridyl-transferase reaction, which is catalyzed by the 28S
rRNA of the large subunit. Peptide bond between the now-adjacent first and second
amino acids is formed that connects one ami.no acid to another, c. g., the methionine

1 The requirement for hydrolysis of GTP before cEF-1 is released from the ribosome is the
rate-limiting step in elongation and provides a time for proofreading of the codon-anticodon
pairing before the peptide bond forms.
3.6. Translation: Prolan Synthesis 149

from tlx first tRNA is transferred onto the amino acid of the second tRNA in the A
site (Fig. 3.32).

Fig. 3.32. The scheme of eukaryotic trail sialion elongation. The initiating methionyl tRNA occupies
the P site, leaving an empty A site. The second aminoacyl tRNA (e.g. glycyl tRNA) is Brought to
the A site By eEF-1 (complexed with GTP). Following GTP hytiroiyas, eEFl {complexed wilh GDP)
leaves ine ribosome with second tRNA {glycyl ffiNA) inserted into the A site. A peptide bond is then
formed, resulting in the transfer of methionine to the aminoacyi tRNA at the A site. The ribosome
then moves three nucleotides along the mRNA. This movement translocates the Mei-Gly tRNA to the
P site and the uncharged tRNA to me E site, leaving an empty A site ready for addition of the next
amino acid. Translocation is mediated by eEF-2 coupled to GTP hydrolyzis. This process, elongation
and translocation, is repeated until all trie amino acids have been added and me elongation complex
reaches the termination codon

After rhe peptide bond is formed, the ribosome translocates again, thus causing
the uncharged tRNA io occupy the E site. The uncharged tRNA is then released to
the cytoplasm (Fig. 3.32) to pick up another amino acid. In addition, the A site is
now empty and ready to accept the next aminoacyl-tRNA.

Termination of Translation
Termination occurs when a stop codon in the mRNA (UAA< UAG, or UGA) enters
the A site (Fig. 3.33). No tRNAs recognize these codons; instead they arc recognized
by proteins called release factors (RF) (Table 3.9). The polypeptide is released, the
ribosome disassembles back into its two independent subunits and translation is
terminated.
150 Chapter 3. Sene expression and protein synthesis

mRNA can be translated simultaneously by more than one ribosome in both

prokaryotic and eukaryotic cells (Fig. 3.34). Once one ribosome has moved away
from the initiation site, another can bind to the mRNA and start the synthesis of
a new polypeptide chain. Usually. mRNA is translated by a group of ribosomes,
spaced at intervals of — 100 to 200 nucleotides, forming a polyribosome or polysome.
Each ribosome within the polysome functions independently to synthesize a separate
polypeptide chain.

Figi 3.31 The scheme off eukaryotic translation termination. Termination occurs when one of the three
slop codons reaches ine A site. Slop codons are recognized Dy RF, whicn fit neatly into ine P site

Fig 3.34. Diagrammatic representation of polysomes

After many ribosomes have completed translation, the mRNA is degraded so the
nucleotides can be reused in another transcription reaction.

Translational Control in Disease

Translation is tightly controlled to ensure that the correct proteins arc synthesized
at the appropriate time and in the quantities needed by the cell Translational control
is crucial in the regulation of gone expression (discussed in Ch. 3.10). Deregulation
of protein translation control is associated with a wide range of different diseases
including certain cancers and metabolic disorders.
3.7. Protein Posttransiational Modifications 151

hi many cancers, the amounts ofc IPs arc increased. For example, there i.s a strong
relationship between levels of ex pression and cancer for cl F4E, whose overexpression
causes malignant transformation of human cells. The transforming activity of'clF4E
can be explained by its ability to promote translation of a subset of mRNAs encoding
proteins involved in regulating growth, proliferation, and apoptosis, most likely in a
combinatorial fashion. Translational control plays a role in learning and memory; it
is implicated in certain neurological disorders, such as fragile X mental retardation
(FMR) syndrome. The disorder is caused by changes in the expression level of
FMR protein (FMRP), which is mutated or produced in reduced amounts in this
disease. FM RP is an RNA-binding protein, which normally inhibits the translation
of mRNAs whose products have critical roles in synaptic plasticity. Mutation or
reduced expression of FM RP results in excessive synaptic plasticity leading to FMR
disorder.

3 J. PROTEIN POSTTRANSLATIONAL MODIFICATIONS

During and after translation, individual amino acids of a polypeptide chain, may
be chemically modified through posttransiational modifications (PTM) (Table 3.IO).
Such, mod ill cations come in a wide variety of types, and arc mostly catalyzed by
enzymes that recognize specific target sequences in. specific proteins. Protein PT Ms
can be reversible or non-reversible depending on the nature of the modification. There
arc two broad categories of PTM:
> covalent additions of some chemical group, c.g.. phosphorylation, acylation,
alkylation, glycosylation;
► covalent cleavage of peptide backbones by either proteases or, less commonly,
autocatalysis.
Attachment of specific chemical groups to amino acid side chains in protein
PTM can be enzymatic or nonenzymatic. Examples of enzymatic PTMs
include phosphorylation, glycosylation1,2 acetylation, methylation, sumoylation,
palmiLoylation,ctc. Nonenzymatic PTMs include glycation1, nil rosy lai ion, oxidation/
reduction, acetylation, and succi nation.
Some of the most common modifications arc methylation that is mediated by
methyl transferases. One-carbon methyl groups arc added to nitrogen or oxygen
(N- and O-methylaLion, respectively) on amino acid side chains. While N - methylation
is irreversible, O-meihylaiion is potentially reversible. The transfer of methyl groups
from .S-adenosyl methionine (discussed in Ch. 8) to histones is catalyzed by histone
methyl transferases. Methylal ion regulates ifgene transcription is activated or repressed,
thus leading to different biological outcomes. Phosphorylation is the most common
PTM mediated by kinases. It is a reversible addition of a phosphoryl group from
ATP to serine, threonine, or tyrosine residues. Phosphorylation plays critical roles
in the regulation of many cellular processes including cell cycle, growth, apoptosis
and signal transduction pathways. Phosphorylation alters the structural conformation
of a protein, causing it to become activated, deactivated, or modifying its function.

1 Glycosylation is enzymatic conjugation with carbohydrates.

2 Glycation is nonenzymatic conjugation with carbohydrates.
152 Chapter 3. Sene expression and protein synthesis

Table 3.10. Example of poslfranslational modifications, meir target amino acid residue!s|- and tne
enzyme(s) or proteins involved

Phosphorylation Tyrosine, serine, Kinases, phosphatases

threonine

Glycosylation N-linked Asparagine Glycosyltransferases, deglycosylases

Glycosylation O-linked Serine/threonine Glycosyltran sferases. deglycosylases

Acetylation Lysine Acetyltransferases, deacetylases

Methylation Lysine, arginine Methyltransferases, demethylases

UbiquiEination Lysine Ubiquitin-activating enzymes, ubiquirin-conjugatmg

enzymes, ubiquitin ligases, deubiquitinases

Sumoylation Lysine Ubiquitin-activating enzymes, ubiquilin-conjugaEing

enzymes, ubiquitin ligases, deubiquitinases

Myristoylation Glycine N- My ristoyltran sferases

Prenylation Cysteine Farnesyitransferases, geranyl geranyttran sferases

Palmitoylation Cysteine OHHC protein acyltransferases, acyl-protein

thioesterases

Sulfation Tyrosine Sulfatases. de sulfatases

S-Nrtrosylation Cysteine, mesh io nine

Glycation Lysine

Nitration Tyrosine Denitrases

Chlorination Tyrosine Myeloperoxidases

Oxidation/red notion Cysteine Peroxidases, oxidases, glutathione, thioredoxin

Carbonylation Lysine, proline,

arginine, Ehreonine

Ubiqu id nation is another PTM me dialed by ubiquitin protein. Ubiquitinal ion is

a reversible modification. Single ubiquitin (8 kl)a), or multiple ubiquitins, arc
attached to the N-terminus of target proteins via the C-terminal glycine of ubiquitin.
Ubiquitination is involved in the regulation of both membrane trafficking and protein
degradation.
PT Ms play a fundamental role in regulating the folding of proteins, their targeting
to specific subccllular compartments, their interaction with ligands or other proteins,
and their functional state, such as catalytic activity in the case of enzymes or the
signaling function of proteins involved in signal transduction pathways.
Many human diseases arc associated with PTM deregulation. For example,
deregulation of methylation is observed in colorectal cancer. Deregulation of
phosphorylation in a variety of proteins that modulate important signal transduction
pathways is associated with the development of several cancers, including breast
and ovarian cancers. Ncurodegencrativc and other neurological disorders, c.g.,
Rubinstein-Taybi syndrome and amyotrophic lateral sclerosis, are characterized by
3.8. DNA, RNA ana Protein Synthesis Inhibitors 153

deregulated histone acetylation. Another example of PTM deregulation is observed in

HlV-infcctcd cells. HIV has accessory proteins that hijack the ubiquitination pathway,
which modifes proteins with ubiquitin. This subsequently leads to the deregulation of
cellular protein PTM.

3.8. ONA, RNA AND PROTEIN SYNTHESIS INHIBITORS

Replication, transcription, and translation arc complex processes and arc favorite
targets for inhibition by toxins, poisons, and antibiotics.
► Antibiotic is the substances produced by bacteria or fungi which inhibits
the growth of other organisms. The majority of the antibiotics interfere with
bacterial protein synthesis and arc harmless to higher organisms;
► Poison, in biochemistry, is a substance, natural or synthetic, that causes damage
to living tissues and has an injurious or fatal effect on the body, whether it is
ingested , inhaled, or absorbed or injected through the skin:
► A toxin is a poisonous substance produced within living cells or organisms.

Antitumor Antibiotics
Antitumor antibiotics arc not the same as antibiotics used to treat bacterial
infections. Antitumor antibiotics arc a type of anticancer drugs that block cell growth
by interfering with DNA inside cells. This slows or slops cancer cells from growing and
keeps them from multiplying.
Anthracyclines are antitumor antibiotics initially developed from compounds
produced by This class includes daunorubicin. doxorubicin, idarubicin,
and epirubicin as well as liposomal formulations of daunorubicin and doxorubicin.
They exon their cytotoxic effects through several proposed mechanisms:
► intercalation between DNA base pairs, resulting in disrupting DNA and RNA.
synthesis;
► inhibition of topoisomerase II. resulting in double-strand DNA breaks during
DNA replication.
Dactinomycin, also known as actinomycin D, is antitumor antibiotic used to treat
a number of types of cancer. It binds to DNA and inhibits RNA synthesis resulting in
impaired mRNA production.
Bleomycin is an antitumor antibiotic and acts by induction of DNA strand breaks
leading to inhibition of RNA synthesis.
Mitomycin C is an antitumor antibiotic and acts as a double-stranded DNA
alkylating agent. Mitomycin C covalently crosslinks DNA, inhibiting DNA synthesis
and cell proliferation.
Puromycin is an aminonudcosidc antibiotic that possesses antitumor activity.
Puromycin acts as a protein synthesis inhibitor. It does not inhibit the enzymatic
process, but instead competes by acting as an analog of the 3'-terminal end of
ami noacyl-1 RNA, disrupting synthesis and causing premature chain termination.
Vinblastine is an antitumor alkaloid isolated from plant Vinblastine
binds to the microtubular proteins of the mitotic spindle , leading to crystallization of
the microtubule and mitotic arrest or cell death.
154 Chapter 3. Sene expression and protein synthesis

Inhibition of Bacterial Protein Synthesis by Antibiotics

Several drugs inhibit protein synthesis in bacteria without significantly interfering
with protein synthesis in human cells. The selectivity is due to the differences between
bacterial and human ribosomes. The antibiotics are classified based on the mechanism
□faction:
► chloramphenicol, erythromycin, clindamycin, and linczolid act on the SOS
subunit (Table 3.11):
► tetracyclines and aminoglycosides act on the SOS subunit (Table 3.11).

Table 3.11. The moite of action of antibiotics that inhibit protein synthesis

Aminoglycosides 30S Blocks functioning of initiation complex and causes misreading of mRNA
Tetracyclines 30S Blocks tRNA binding to ribosome
Chloramphenicol 50S Blocks peptidyltransferase
Macrotides SOS Blocks translocation
Clindamycin SOS Blocks peptide bond formation
Linezolid 50S Blocks early step in ribosome formation
Telrthromyciit SOS Same as macrolides
Streptogramins SOS Causes premature release of peptide chain

Inhibition of Template Biosynthesis by Poisons and Toxins

a-Araanitm is a highly toxic cyclic peptide of eight amino acids found in mush­
rooms (genus /ImoM a-Amanitin is a. selective inhibitor of RNA polymerase II
and llh
► RNA polymerase II is the most sensitive polymerase:
► RNA polymerase III is a hundred-fold less sensitive than RNA polymerase 11.
Amanitin is adsorbed through the intestinal epithelium and binds weakly to serum
proteins. The liver is the principal organ affected, as it is the first organ encountered
after absorption in the gastrointestinal tract. Once in the liver, it is transported by a
nonspecific transport system into hepatocytes, producing an extensive centrilobular
necrosis. About 60% of absorbed a-amanitin is excreted into the bile and is returned
to the liver via the entcrohepatic circulation.
(i-amanilin traps a conformation of the RNA polymerase 11 enzyme that prevents
nucleotide incorporation and translocation of the transcript. This leads to a progressive
decrease in mRNA production, deficiency of protein synthesis, and cell death.
Ricin is a poison found naturally in castor beans ( Jfenws Ricin
inhibits protein synthesis and stimulates cells to undergo programmed cell death
(apoptosis).
Diphtheria toxin (DT) is produced by toxigenic strains of C and is
responsible for the symptoms of diphtheria. DT belongs to bifunctional A-1? toxins.
The A subunit of diphtheria toxin catalyzes ADR ribosylationofthe EF-2. inactivating
EF-2, and turning off protein synthesis. DT is able to inhibit protein synthesis of all
eukarvoiic cells.
3.8. ONA, RNA ana Protein Synthesis Inhibitors 155

Cyl in dro sperm opsin is a cyanolox in produced by a variety of freshwater

cyanobacteria. Cylindrospermopsin accumulates in liver over lime, binds to DNA,
causes DNA fracmen ration, and inhibits protein synthesis.
Phyllanthoside is a glycoside isolated from the roots of the Central American tree
Phy/ldnfJjMi acumtna/us Vafcf and nagilactone C is a toxic substance from /Wgco/jpu'.s
werii/bfiwjj, both inhibit protein synthesis exclusively in eukaryotic cells.
Interferons (IFNs) are cytokines, a group of signaling proteins, made and
released by host cells in response to the presence ofvirus/viruses or other pathogens.
In addition to their antiviral properties, IFNs also exhibit antiproliferative,
immunomodulatory; and many other activities. IFNs do not have direct antiviral
activity, but rather exert antiviral effects by inducing the production of more than
two dozen effector proteins in exposed cells:
> in response to interferon, cells produce enzymes that reduce protein synthesis
within the cell:
► inhibited protein synthesis impairs both virus replication and infected host
cells.

Questions and situational problems

1. In the diagram, label the three tRNA sites, codonsand anticodons, peptide and
mRNA. List the sequence of events that occur when the incoming tRNA sets
into its binding site. Redraw the diagram as it will appear immediately after the
next peptide bond is formed.
2. Working with an animal celt culture system, a researcher created random
point mutations in the genes for the RNA polymerases. Individual cells with
RNA polymerase mutations were isolated and used to generate a cell line
that expressed that particular mutation. There was only one problem; the
researcher did not know which RNA polymerase was affected in each, of the
cell lines. To help resolve this question, the researcher analyzed the levels of
different RNAs expressed in each of the cell lines. The obtained results arc
presented below:
156 Chapter 3. Sene expression and protein synthesis

Tpnstoe
Unmutated cells 100 100 100

Cell line 1 100 98 40

Cell line 2 20 100 95

Cell line 3 43 100

Which type of RNA polymerase {I, II. or 111) appears to be mutated in each one
of the cell lines? Explain.
3. ?! particular gene codes for a mature mRNA contains 900 bases, which is
translated into a 40 kDa protein. A mutant form of the gene created by a
single point mutation yields a 30-base mRNA yielding a short peptide with
nonfunctional enzymatic activity. Why is the obtained product nonfunctional?
To answer die question please:
1) draw a diagram of protein synthesis without mutation;
2) explain what is the genetic code and list the properties of the genetic code;
3) list the sequence of events that occur when a mutated mRNA is translated at
the IOth amino acid;
4) redraw the diagram as it will appear immediately after the peptide bond is
formed between the 9th and I Oth amino acids.
4. Doxorubicin interacts with DNA by intercalation and inhibition of
macromolecular biosynthesis. It blocks topoisomerase II. When this drug can
be used? To answer the question please:
1) draw a diagram of macromolecular biosynthesis that is inhibited by the drug:
2) how docs doxorubicin affect the macromolecular biosynthesis?
5. A schematic diagram shows the translation process at the stage of incorporation
of the 6th amino acid into the growing p-chain of Hb. Determine what amino
acid will occupy the 6th position in p-protomers of Mb. To answer the question
please:
1) mark the 5’ and 3’ ends;
2) redraw the diagram as it will appear immediately after the peptide bond is
formed between 5th and 6th amino acids:
3} what type of Hb has this amino acid sequence?
Vai-Met

\
mRNA

/
6. Antibiotics are used in the treatment and prevention of bacterial infections.
Tetracyclines inhibit protein synthesis by binding to the ribosomal small subunit
(3 OS) of prokaryotes and blocking the ami noacyl-tRNA binding to A site. Why
docs tetracycline only affect bacteria? To answer the question, please explain:
3.8. ONA, RNA ana Protein Synthesis Inhibitors 157

I ) what is ribosome?
2) what is the difference between prokaryotic and eukaryotic ribosomes?
3) what is the function of ribosome in a cell?
7. Determine which amino acid should be attached to tRNAs with the following
anticodons:
1) 5’-UAC-3’;
2) 5’-GAU-3’;
3) 5’-AUU-3’;
4) 5’-AUG-3’.
8. Design mRNA sequence of a protein fragment: N-Ala-Pro-Mct-Thr-C.
9. In sickle cell hemoglobin, there is a Vai residue at position 6 of the chain,
instead of the Glu residue found in this position in. normal 14 bA. Can you
predict what change look place in the DNA codon for glutamate lo account for
its replacement by valine?
10. A portion of an mRNA molecule has the sequence
5,-AUGCCACGAGUUGAC-3\ What amino acid sequence does this code
for? To answer the question please:
1) explain what is the genetic code and list the properties of the genetic code:
2) draw a diagram of protein synthesis;
3) determine which iRNA should be attached to the mRNA:
4) what is the anticodon for the very first iRNA that will attach to mRNA?
11. The peptide hormone oxytocin contains 9 amino acids. What is the minimum
number of nucleotides needed to code for this peptide?
12. A portion of the coding strand of a gene was found lo have the sequence
S'-ATCjAGCXjACTrTCCjCCCATTA-S'. A mutation occurred in the gene,
making the sequence 5'-ATGAGCGACCTTCGCCCATTA-3\ What effect
would the mutation have on the amino acid sequence of the protein obtained
from this mutated gene? To answer the question please:
1) explain what the genetic code is;
2) identify what mutation occurred;
3) what amino acid sequence docs the non-mutated and mutated gene code for?
13. Glycosylation is the addition of to the protein.
A. Carbohydrate;
B Upid;
C. Fat;
D. Minerals.
14. Which of the following is not an example of post-translational modification?
A. Polyadenylation:
B. Myristoylation:
C. Alkylation;
D. Ubiquili nation.
15. Fill in
In a ribosome, the formation of the peptide bonds of the new peptide chain
occurs in the ________subunit, whereas matching the codons of the mRNA are
exposed on the surface of the subunit. During the peptide elongation
stage of translation, each incoming ammoacyl-lRNA binds lo the.
158 Chapter 3. Sene expression and protein synthesis

-site of the ribosome, whereas the growing peptide chain is held on the t RNA in
the -site.
16. Antibiotics arc used in the treatment and prevention of bacterial infections.
Macrolides, c.g., erythromycin, azytromycin, and clarithromycin, bind to
the 23S rRNA component of the SOS subunit of ribosome and interfere with
the assembly of SOS subunits. Elongation is prematurely terminated. Why do
macrolides on tv affect bacteria? To answer the question please explain:
I} what is ribosome;
2) what is the difference between prokaryotic and eukaryotic ribosomes;
3) what is the function of ribosome in a cell?
4} draw a scheme of a process that is inhibited by macrolides.
17. A scheme of eukaryotic ribosome is shown below. Redraw the scheme in the
copybook and fill in the boxes with the question mark. What is the function of
ribosome in a cell? What arc the three functional sites in the ribosome?

IS. A scheme of eukaryotic translation is shown below. Indicate template,

substrates, sources of energy that arc involved in the process of translation.
Indicate the codon and anticodon for the Mel-1RNA that is attached to
mRNA.
Large ribosomal
subunit
P site

Small
ribosomal subunit
3.9. The Regulation of Gene Expression 159

3.9. THE REGULATION OF GENE EXPRESSION

In a multicellular organisms, nearly all cells have die same DNA. A cell typically
expresses only a fraction of its genes, and die different types of cells in a multicellular
organisms arise because different sets of genes are expressed. Specific functions of
different cell types arc therefore generated through differential gene regulation. In
single-celled organisms, c.g., prokaryotes, gene regulation primarily ensures that a
cell’s resources arc not wasted making proteins that the cell docs not need at that
time. Gene regulation is the process of controlling which genes in a. cell’s DMA arc
expressed. The regulation occurs in both prokaryotes and eukaryotes, but it is far more
complex in eukaryotes.

The Regulation of Gene Expression in Prokaryotes

Some of the proteins arc needed routinely, while others arc needed only under
certain circumstances. Cells do not express all the genes in the genome all the time.
Cell only expresses the genes that fit its current needs. For Esdicridwa cd/i\ (/.' co/z), as
for most cells, glucose is the most readily metabolized sugar, but glucose is not always
available. /’ co// can use cither glucose, which is a monosaccharide, or lactose, which
is a di saccharide. However, lactose has to be hydrolyzed first. The bacterium prefers
to use glucose when it can. Genes for lactose utilization arc activated once £. ro/t colls
run out of glucose and sense the presence of lactose or a similar compound. Cells
respond to changes in the envi ronment by differentially controlling genes.
The DNA of prokaryotes is organized into a circular chromosome supc reoiled in
the nucleoid region of the cell cytoplasm. Prokaryotic transcription and translation
occur simultaneously in the cytoplasm, and regulation occurs at the transcriptional
level.

Prokaryotic Gene Regulation: Structure of an Operon

Proteins that are required fora specific function arc encoded together in blocks
called operons (Fig. 3.35). They arc transcribed together under the control of a single
promoter. The promoter regulates the expression of the genes in a manner all-or-none,
because the products of these genes will cither all be needed al the same time, or none
will be needed. The opcron’s regulatory region includes both the promoter and the
operator.
Regulatory molecules that can affect the expression of opcrons arc co-repressors,
activators, and inducers:
> repressors — proteins that suppress transcription of a gone in response to an
external stimulus:
► activators — proteins that increase the transcription of a gene in response to an
external stimulus:
* inducers are small molecules that cither activate or repress transcription
depending on the needs of the cell and the availability of substrate. They help
speed up or slow down «on* or «off* by binding to a repressor or activator.
160 Chapter 3. Sene expression and protein synthesis

Operon
P^moiwn^tor . Smictogl panes
Structural genes
Regulatory gene
DNAfp
* - *■ — ■

■ , ■, -,B,c
Transcription |

mRNA

Illi
Translation

O O A O
Protein Protein Protein Protein
A B C D

Fig. 3.35. Structure of an operon Three basic components of an operon: promoter — upstream
sequence to which RNA polymerase binds; operator — segment of ONA to which a
repressor protein binds (inhibits transcription by obstructing RM A polymerase); structural
genes — genes that are collectively regulated by the operon. Structural genes encode
products that serve as enzymes, and regulatory genes encode products that regulate gene
expression

If a repressor binds to the operator then the structural genes will not be
transcribed. Alternatively, activators may bind to the regulatory region, enhancing
transcription.

Prokaryotic Gene Regulation: kjclndudble Operon

The lactose ffocj operon in £ co// is an. example of an inducible operon
(Fig. 3.36). The foe operon consists of throe genes, focZ, focKand foed. each, involved
in processing the sugar lactose. LacZ is the gene for the enzyme [J-galaclosidasc.
P-galactosidase hydrolyzes lactose into glucose and galactose. Together these gene
products act to import lactose into celts and break it down for use as a food source.
Operator and terminator arc regulatory sequences to ensure coordinated regulation
of the foe operon:
► the operator is a special DNA sequence located between the promoter sequence
and the structural genes that enables repression of the entire foe operon , following
binding by the repressor-inhibitor (lacf) protein;
► the terminator is DNA sequence that marks the end of an operon in genomic
DNA during transcription.
The regulatory gene foe/ encodes a lac repressor protein, which can bind to the
operator of the foe operon and inhibit transcription of the foe operon. The lac repressor
acts as a lactose sensor. It normally blocks transcription of the operon, but stops acting
as a repressor when lactose is present.
3.9. The Regulation of Gene Expression 161

For the foe operon io be expressed, lactose must be present. Otherwise, it is

useless to synthesize the enzymes to process lactose. In the absence of lactose, the
/de repressor is bound to the operator region of the /oc operon, physically preventing
RNA polymerase front transcribing the structural genes. Removal of the repressor in
the presence of lactose allows RNA polymerase to move through the operator region
and begin transcription of the /<?c structural genes.

^lac Operon d
Struc-tLFai genes

FTomoler ric-gj-ai Operator [ lacZ lacY | lacA Terminator

Trans rapti on

Lactose binds to tte

inriibilw and prewenis | mHNA
i tinding 1o lhe
operator, thereby
TransLslion avowing transoripbon

Binds to ifte
operator to inhibit
bansenption

lad regulator
>Enzymes La import lactose
pro tetri and break it down

Fig. 3.36. The tec operon in £ rafi All of the opera's lactose metabolism genes (/acZ, tecYano tecA)
are vacated next to each other downstream of a single promoter. This promoter serves as a recognition
site for tne transcriptional machinery of the RNA polymerase complex. All genes in an operon are
transcribed into a single mRNA molecule, wnich is subsequently translated into individual protein
products. A tec/ regulator gene with its promoter is found just outside the lac operon. Tne regulatory
gene lad produces a tec repressor protein. When lactose is absent, the lac repressor protein binds to
tne operator, blocking lhe RNA polymerase from transcribing the tec structural genes. When lactose
is available, a lactose molecule binds tne tac repressor protein, preventing the repressor from binding
to tne operator sequence, and the genes are transcribed

To quickly adapt to environmental changes co/i evolves mechanism to turn

lactose-metabolizing genes on or oil as a group. /■'. cofi uses this system to tightly
control the genes required for the use of lactose.
With a few exceptions (e/egarcs and related nematodes), eukaryotic
genomes do not have genes arranged in opcrons. Instead, eukaryotic genes that arc
co-regulated tend to have the same DNA regulatory element sequence associated
with each gene, even if those genes arc located on completely different chromosomes.
162 Chapter 3. Sene expression and protein synthesis

j.c., the same transcriptional activator or repressor can regulate transcription of every
single gene that has that particular I)NA regulatory element associated with it.

The Regulation of Gene Expression in Eukaryotes

During cellular development and diflcrcntjatjon. a number of mechanisms
precisely determine when, how and in what amounts each protein must be
synthesized. These mechanisms, collectively known as regulation of gene expression,
arc also essential for cell response to changing environmental conditionsand defense
against pathogens.

Chromosomal DNA and its Packaging in the Chromatin Fiber

Each human cell contains approximately two meters of DNA in its nucleus.
This DNA must lit into a nucleus with a diameter of only about 6pm. The task of
packaging DNA is accomplished by specialized proteins that bind to and fold the DNA
into a very compact and tight structure. These proteins arc called histones, and the
resulting DNA-protein complex is called chromatin, importantly, although the DNA
is very tightly folded, it is compacted in a way that allows it to easily become available
to the many enzymes in the cell that replicate it, repair it. and use its genes to produce
proteins.
Histones are a family of small, positively charged proteins termed Hl, H2A,
H2B, H.k and H4. DNA has a negative charge, due to the phosphate groups in
its phosphate-sugar backbone. The positively-charged histones strongly adhere
to DNA and form nucleosomes. In the nucleosome, about 200 bp of DNA arc
wrapped around a histone octamer core («octamcr&), which, consists of 8 histone
proteins (two each of histones H2A. H2B. H3, and H4). The remaining bases link
to the next nucleosome and are called linker DNA (Fig. 3.37 A). Such a structure
allows the DNA to be condensed into a smaller volume (Fig. 3.37 H) and resembles
beads on a siring when extended and viewed under a microscope (Fig. 3.37 C).
Interestingly, the wrapping of DNA in the nucleosome permits two regions that arc
apart in the DNA primary sequence to be brought into close proximity. Thus, two
sequences that arc apart can interact with the same regulatory protein to control
gene expression.
3.9. The Regulation of Gene Expression 163

A B
Octamer of core nistonos:
H2A, H2Br H3. H4 (each ons x2)

Histone Core

<------------Core DNA
Linker

Nucleosome

Nucleosome core

F------------- H
0.05 gm

Fig. 3.3T Nucleosome structure. |A) Nucleosome structure is tne basic unit of DNA packaging. It is
formed through trie interaction between DNA and nisione proteins. A segment of ONA is wound around
a core of 8 histone proteins (two each of histones H2A, H2B, H3: and H4i. The «taiis* of these histone
proteins stick out, where they can be modified by a number of different nistone acetyltransferases,
methyitransferases, and other epigenetic enzymes. Many of these enzymes modify very specific sites.
(B) The core DNA forms two loops around the octamer. The DNA that is between each histone octamer
is called the linker DNA. and can vary in length from 8 to 114 base pairs. This variation is species
specific, but variation in linker DNA length has also been associated with tne developmental stage of
the organism or specific regions of the genome. (C) Electron micrograph of chromatin: the beads on
a string

At a higher level of ch romat in organization , the chain of nucleosomes is compacted

further io form chromatin fiber, which is, in turn, folded into a higher-order
chromatin loop structure called a chromosome (Fig. 3.38).
164 Chapter 3. Sene expression and protein synthesis

Short region of
Dima double helix

“Beads on a string"
1 nm
form of chromatin

30-nm cnromatin
fibre of packed out
nucleosomes

Section of
chromasome in an 300 nm
extended form

Condensed section
700 nm
of chromosome

Centromere

Enti/e mitotic
1.400 nm
chromosome

fig 3.38. Distinct levels of chromatin organization Mote me folding of me - beads on the string
structures into 30 nm fiber and further folding into higher-order loop structures
3.9. The Regulation of Gene Expression 1B5

The Mechanisms of Regulation of Gene Expression in Eukaryotes or Why Every Cell

does not Express AU of Its Genes
In a multicellular organisms, gene regulation controls which genes in a cell’s
I)NA arc expressed. Unlike prokaryotic cells, eukaryotic cells can regulate gene
expression at many different levels.
»• Chromatin accessibility;
> DNA methylation;
»• Gene rearrangement;
> Transcription;
* Posttranscriptional RNA processing;
* RNA stability;
> Translation.

The Regulation of Chromatin Accessibility for Transcription

The first level of regulation of gene expression in eukaryotes occurs even before
transcription is initiated and is based on selective access to DMA through reversible
rearrangement of chromatin structure. Chromatin in a cell nucleus exists in two
distinct states of compaction: highly condensed, tightly packed heterochromatin and
less condensed, «opcn» euchromatin. The lightly packed structure of nucleosomes
in heterochromatin hinders promoter access and initiation of transcription.
Consequently, genes in the heterochromatin are transcriptionally silent, whereas
those in the euchromatin are transcriptionally active, serving as a template for mRNA
synthesis. During cellular development and differential ion, various chromatin
regions undergo transition from an open to a. condensed state or vice-versa leading
to cither selective gene expression or repression. The enzyme-assisted process of
heterochromatin conversion to euchromatin is called chromatin remodeling. This
process involves nucleosome displacement from specific DNA regions making them
accessible to transcription machinery and occurs through two mechanisms.
The first mechanism is covalent post-translational histone modification by specific
enzymes, c.g., histone acetyl transferases (HATs), histone deacetylases (HDACs),
rnclhy It ransfc rases, kinases, ubiquitin ligases, etc. Different posl-transIaiionaJ
modifications affect the binding affinity between histones and DNA in different
ways, loosening or tightening the condensed DNA wrapped around histones. For
example, histone acetylation by HATs removes a positive charge from the r-a mi no
group of lysine in the histone tail, thereby reducing electrostatic interactions
between the histones and the negatively charged DNA, making it easier for DNA to
unwind from the histones. HDACs can reverse this process by removing the acctyi
groups. Mono-, bi- or tri- methylation of certain lysine residues in the histone tail
by methyl t ransfc rases correlates with transcriptional activation or repression. The
complex and varied role of histone modifications in chromatin remodeling has
even led to the proposal of a histone code hypothesis, according to which histone
modifications act sequentially or in combination to form a code that determines
chromatin structure and, consequently, regulates specific gone expression programs.
The second mechanism involves ATP-dependent chromatin remodeling complexes,
which utilize energy from ATP hydrolyzis to reposition nucleosomes along the DNA,
166 Chapter 3. Sene expression and protein synthesis

expel histones away from DNA or facilitate the exchange of histone variants, thus
creating nucleosome-free regions of DNA for activation of transcription.

The Regulation through DNA Methylation

A chemical tag called a methyl group (CH,) can be added to the 5 position of
cytosine residues in DNA to produce 5-methylcytosinc. This process occurs in GC-
rich sequences (called GC-islands)., which arc often near or in the promoter region
of a gene. DNA methylation in these regions play's an important role in regulating
gene expression during embryonic development and typically acts to repress gene
transcription. For example, DNA methylation is involved in the inactivation of one
of the two copies of the X chromosome present in female mammals. This prevents
females from having twice as many gene products encoded by the X chromosome as
males.

The Regulation through Gene Rearrangement

Gene rearrangement represents a mode of gene regulation in which segments
of DNA arc shuffled in various ways, giving rise to different proteins. Gene
rearrangement is best studied for its role in the production of key proteins of the
immune system, such as immunoglobulins, also known as antibodies, and T-cell
receptors. Although every human organism is capable of producing a vast spectrum of
different antibodies, each B lymphocyte produces only a single type of antibody. This
is achieved through site-specific recombination during I? lymphocyte development, a
process of DNA breakage and reunion that rearranges immunoglobulin gene regions
(V, D and J) creating unique gene variants in individual B-lymphocytes. As a result,
the population of approximately 1012 B-lymphocytes in the human body includes cells
capable of producing antibodies against a vast variety of foreign antigens. When the
immune system encounters such an antigen, the B-lymphocyte that can bind to that
antigen is stimulated to proliferate and to produce antibodies against the antigen. A
similar gene rearrangement strategy allows T-cell receptors to achieve a degree of
diversity that matches that found in. antibodies.
Gene duplication or deletion represents yet another means by which gene
expression can be affected prior to initiation of transcription. However, in mammals,
such means of amplifying or abolishing gene expression arc found, with very few
exceptions (such as certain types of placental and neural stem cells}, in cells damaged
by disease such as cancer.

The Regulation at the Level of Transcripti on

The accurate and robust regulation of gene transcription is at the heart of cell
differentiation during development, determining into what cell type a stem cell
ultimately differentiates into. The correct regulation of gene transcription is also
essential for normal cell function and thus represents a corner stone for complex
biological life.

Transcription Factors
Much of the information required for regulation of gene expression at the
transcriptional level is encoded by enhancers, which arc short (-100-1000 bp)
3.9. The Regulation of Gene Expression 1&7

noncod ing DNA sequences located al various distances from the respective promoter.
Enhancers can be anywhere in the genome: upstream or downstream of the promoter,
within gene introns, in distal intcrgcnic regions or even within gene exons. Such
remarkable location flexibility is achieved through *DNA looping* which brings
together distal enhancers and promoters to regulate gene expression (Pig. 3.39). On
the nucleotide sequence level, enhancers contain clusters of short consensus binding
motifs recognized in a sequence-specific manner by regulatory proteins called specific
transcription factors (TFs, not to be confused with general transcription factors, see
Ch. 3.4).

Fig. 3.39 The regulation ol gene transcription. RNA polymerase ll (RNA Pol IL. light green) and its
associated general transcription factors bind to me promoter (green). The muitiproteirr Mediator
complex (Med+Cdkfl {cell division protein kinase 8) stabilizes promoter/enhancer ONA loops Dy
physically bridging transcription factors bound at enhancers (purple) with the RNA polymerase ll
transcription machinery. thereby coordinating transcription initiation events. Mediator also stimulates
deposition of the ring-shaped cohesin complex off genes by sliding tne complexes along DNA

Consensus binding motifs of many TFs represent palindromes, i.c., strings

with a 5’ to 3? sequence that is identical to the 5’ to 31 sequence of the reverse
complimentary strand. For example, the consensus binding motif of TFs belonging
to the ATF/CREB family1 TGACGTCA is a palindrome (Fig. 3.40 A). Generally,
enhancer activation requires the binding of multiple TFs and such cooperative
binding is believed to play an important role in overcoming the energetic barrier of
nucleosome eviction. Multiple TFs can be recruited to enhancers not only through
underlying consensus binding motifs but also through protein-protein interactions.
The ability of TFs to activate transcription is also dependent on the recruitment
of coactivators, which themselves lack DNA-binding domains, but act through

1 The ATF/CREB family is a group of transcription factors consisting of different ATFs

(activating transcription factors), CREB (cAMP response element-binding protein).
168 Chapter 3. Sene expression and protein synthesis

modifying and remodeling the chromatin context of enhancers. A large mu Eliprote in

complex, called the Mediator, also functions as a critical transcriptional coaclivator
by transmitting signals from TFs to RNA Polymerase II (Fig. 3.39). TFs can be either
ubiquitous, continuously expressed in many cell types and tissues, or tissue- and
cell type-specific. This, together with TF ability to activate transcription over long
genomic distances facilitates the execution of enormously complex gene expression
programs in different tissues and cell types using a relatively limited set of genes.
Although there are millions of TF consensus binding motifs in. the human genomen
only a fraction of them is actually occupied by TFs and it is not yet fully understood
how TFs select certain consensus motifs among others. The underlying principles of
TF consensus mot if organization (often referred to as enhancer ^grammar*) remain
an area, of active research.

A ATF/CREB consensus binding motif

5’ TGACGTCA3’
Tilt
ill!
3' ACTGCAGT 5’

Palindromic sequence
B

N I J DBD I J TAb 'Jc

Fi^ 3.40. THe cansa&s Oincfing motif of an ATF/CREB3 lnsiiscriplion factor represents an examine
at a palindromic sequence, in which the 6' to 3' base pair sequence is identical on both strands (A).
Domain organization of a typical transcription factor. Abbreviations: DBD. DMA binding domain; TAD.
transactivation domain (Eh

TFs have a modular structure, typically composed of different functionally

distinct domains t Fig. 3.40, B). The first domain, which is always present in a TF,
is the DNA-binding domain (DBD). The function of this domain is to selectively
recognize and interact with its corresponding DNA consensus sequence. Some of
the best-st Li died DBD examples include a zink linger, leucine zipper, he lix-t urn­
helix and helix-loop-helix domains. The second domain, called the trans-activating
domain (TAD), is responsible lor protein-protein interactions with transcriptional
co activators. Some TFs act as receptors binding their respective ligands while in
the cytoplasm before they can relocate into the nucleus. These TFs, called nuclear
receptors, contain additional domains such as Ligand-binding domain (LBD),
dimerization domain and a nuclear localization signal.
Because of their important role in regulating transcription, TFs are implicated
in a vast variety of human diseases. A classic example is the AfKC proto-oncogenc
encoding the MYC transcription factor, which is a <■ master regulator* of cellular
growth and metabolism. Overexpression of c-.WKC leads to the increased expression
of many genes, including those involved in cell proliferation. thereby leading to the
uncontrolled cell growth and formation of cancer.
3.9. The Regulation of Gene Expression 169

The Regulation of Transcription Factors

There arc several ways to regulate transcription factor activity. First, the activity
of a given TF will depend on the a*ailability of its respective eoactivators. The rate
of transcription will increase or decrease in response to coaciivator upregulation or
downregulation. Another means of regulation is to activate or repress transcription of
genes, themselves encoding TFs. For example, a TF may activate transcription of a set
of genes, among which there is a gene encoding a second TF. After its synthesis, this
second TF may activate another set of genes, among which there is a gene encoding a
third TF. etc. Thus, one TF can set off a scries of events that result in the activation of
several other TFs and many different sets of genes.
Another important means of regulating TF activity is by protein post-translational
mod ideations (discussed in Ch. 3.7). Those modifications may include, but arc not
limited to, phosphorylation. acetylation, sumoylalion, and ubiqui Li nation. and
represent an important means for regulating TF activity and availability. Among them,
phosphorylation is particularly important because it is rapid and reversible. Activation
of signaling pathways often triggers bursts of transcription through changes in the
phosphorylation state of specific TFs. In this way, TF phosphorylation links signaling
from cell surface receptors to activation of specific transcriptional programs in the
nucleus. Furthermore. TF phosphorylation also plays a critical role during the cell cycle,
allowing, due to its reversible nature, the repeated resetting of oscillating transcriptional
programs.
Finally, transcription of target genes can be stimulated when specific TFs. such
as nuclear receptors, bind their respective ligands and are translocated from the
cytoplasm into the nucleus (discussed in Ch. 4 and 9). Examples of such transcription
factors arc steroid and thyroid hormone receptors. They control transcription and,
thereby, have effects in all cells within the body. Ail of these receptors arc composed of
a single polypeptide chain that has three distinct domains (Fig. 3.41):
► the N -terminus: in most cases, th is domain is involved in activating or stimulating
transcription by interacting with other components of the transcriptional
machinery. The sequence is highly variable among different receptors:
► DNA binding domain: this domain is responsible for binding of the receptor to
specific sequences of DNA;
* the C-terminus or ligand-binding domain: this domain binds hormone.

NH2- interacts with Dinar TFs DNA binding J Hormone binding - COOH

Fig. 3.41. Three distinct domains of steroid and thyroid hormone receptors

Lipophilic molecules, e.g., steroid hormones enter the cell by simple diffusion
across the plasma membrane. Thyroid a hormones enter the cell by facilitated diffusion
t discussed in Ch. 4). When a hormone binds to a receptor, number of events occurs:
> receptor activation;
»■ activated receptors bind to «hormone response elements*;
► activation or downregulation of transcription from those genes to which the
receptor is bound. Most commonly, receptor binding stimulates transcription.
170 Chapter 3. Sene expression and protein synthesis

Importantly; rhe production of too much or too little steroid or thyroid hormones
causes a number ofclinically important disorders, c. g_, thyroid hormone receptors are an
important regulator in different processes including development, growth, metabolism,
etc. The hyperthyroid condition results from an overproduction of thyroid hormones
resulting in a continual stimulation of thyroid receptors which is detrimental for the
patient.
It has to be noted that TFs often work cooperatively, the binding of one TF to DNA
may enhance the binding of a second TF to a nearby location. TF activity can also be
stimulated or inhibited through direct protein-protein interactions with other TFs.

The Regulation through Posttranscriptional Processing of RNA

After the gene is transcribed (i.c., post transcription), particular exons, portions
of exons or noncoding regions of a gene may be included within or excluded from
the final, processed mRNA. The use of alternative splice sites or sites for addition of
the poly(A) tail (polyadenylation sites) can result in the production of proteins with
different amino acid sequences from the same gene (sec Ch. 3.4). Il is thought that
al least 75% of roughly 30,000 human genes undergo alternative splicing to encode
two or more protein isoforms and errors in this intricate process can lead to many
diseases. For example, a single nucleotide polymorphism (a substitution of one
nucleotide base with another) in exon 12 of the low-density lipoprotein receptor
(LDLR) promotes skipping of this exon. This generates a dysfunctional iso form of
the receptor that is unable to efficiently remove low-density lipoproteins from the
bloodstream, leading to a buildup of cholesterol in arteries (arteriosclerosis).
Another mechanism of posttranscriptional gene regulation is RNA editing.
In this mechanism, bases arc altered, substituted or deleted after the transcript is
synthesized so that the mature mRNA and Lhe corresponding protein sequences
differ from those predicted by the genomic DNA sequence.
In addition to alternative splicing and RNA editing, gene expression can be
dow n regu lated after l ra nsc ript ion t hrough a b iolog ical precess called posit ran seri pt ionol
gene silencing (PTGS) or RNA interference (RNAi). PTGS/RNAi inhibits gene
expression by neutralizing mRNA molecules. Small RNA molecules called mi era RNA
(miRNA} and small interfering RNA (siRNA), which arc complementary to their
mRNA targets, arc central to PTGS/RNAi. These small RNAs can direct specific
RNA-induccd silencing complexes to degrade mRNA. prevent mRNA from being
translated or catalyze DNA or histone methylation to downrcgulatc genes pre-
transc optionally.

The Regulation at the Level of RNA Stability

Although eukaryotic mRNA is it self relatively stable (with half-lives varying be tween
30 minutes and several days), it can be rapidly degraded by cellular ribonucleases.
To prevent such degradation during transport from the nucleus to the cytoplasm, a
poly (A) tail is added to the 3’ end of an mRNA molecule through polyad cnylation
(see Ch. 3.4). The longer the poly(A) tail, the better it protects mRNA from nuclease
attack. Consequently, mRNA with longer poly(A) tail has a longer lifespan, allowing
more protein to be translated from it. As m RNA ages, its poly (A) tail gradually becomes
shorter. The importance of polyadenylation in the regulation of mRNA stability is
3.10; The Effects of Mutations 171

underscored by the fact that a number of diseases, including thrombophilia, spinal

muscular atrophy, and osteoarthritis arc associated with defects in this process.

The Regulation at the Level of Translation

In eukaryotes, gene expression can be regulated at the initiation stage of
translation, via phosphorylation of eIPs (eukaryotic initiation factors, sec Ch. 3.6).
Phosphorylation of elFs affects the recruitment of the mRNA to the 40S ribosomal
subunit, binding of Met-tRNAj, scanning and initiator codon. For example, heme
regulates translation of globin mRNA in reticulocytes (red blood cell precursors) by
preventing phosphorylation of cl F2 by a specific kinase. Reticulocytes lack nuclei and
gene expression in these cells has to be regulated at the level of translation rather than
transcription. Wien heme levels are high, clF2 is not phosphorylated and initiation
of globin synthesis is allowed to proceed. When heme levels are reduced, clF2 is
inactivated by phosphorylation and globin mRNA translation is inhibited.
Besides phosphorylation, post translational modifications such as methylation,
ubiqu it i nation. and glycosylation, may affect protein synthesis. MicroRNAs can
stimulate the degradation of mRNAs or affect protein synthesis directly, thereby
regulating protein synthesis in a cell.

3.10. THE EFFECTS OF MUTATIONS

Mutation is a permanent change in DNA sequence. All cells in the body contain
I) NA: there arc lots of places for mutations to occur. A single base change can create a
devastating genetic disorder or a beneficial adaptation, or it might have no effect. How
do mutations happen (discussed in Ch. 3.3), and how do they influence the future of
a species will be discussed further below.
Somatic mutations (or acquired mutation) can occur in any of the cells of the body
except the germ cells and therefore arc not passed onto offspring. These alterations can
(but do not always) cause cancer or other diseases. Cancer mutations arise in a special
category of genes called proto-oncogenes, many of which regulate cell division Many
of these proto-oncogenes have now been identified and mapped. When mutated, i.c.,
a proto-oncogene becomes a tumor-inducing agent — an oncogene, such cells enter a
stale of uncontrolled division, resulting in a cluster of cells called a tumor.
Germ line mutation (or hereditary mutation) is a gene change in a body’s reproductive
cell that becomes incorporated into the DNA of every cell in the body of the offspring.
Germ Einc mutations arc the ultimate source of congenital diseases.

The Types of Mutations

The types of mutations (Table 3.I2, Fig. 3.42,3.43) include:
► silent mutation causes a change in the sequence of bases in a DNA molecule,
but docs not result in a change in the amino acid sequence of a prole in .This is
possible because the genetic code is degenerate. For example, if the codon AAA
is altered to become AAG, the same amino acid — lysine — will be incorporated
into the peptide chain;
► missense mutation is a change in one DNA base pair that results in the substitution
of one amino acid for another in the protein. Sickle cell anemia is an example of
a disease caused by the mutation:
172 Chapter 3. Sene expression and protein synthesis

Wild type mRNA 5' GCU GGA GCA CCA GGA CAA GAU GGA 3'

Wild-type polypeptide N Ala Gly Ala Pro Gly Gin Asp Gly C

Silent mutation

Note:
Miissense mutation these are all
substitutions

Noneeinse mutation

GCU GGA GCC ACC AGG ACA AGA UGG A Trits on e rs an

Ftameshift mutation
Ala Gly AJa Tti? Am Trip insertion

Fig. 3.42. Ttie scheme illustrating the different types of mutaliarrs. Some mutations do not change
the sequence of amino acids in a protein. Some swap one amino acid tor another. Others introduce
an early stop codon into the sequence causing the protein to De truncated. The red color indicates
mutation. The arrow indicates the insert of one nucleotide

► nonsense mutation occurs when the [3 NA change creates a pre mat urc STOP
codon, which truncates the poly peptide, and usually, nonfunctional protein
product is synthesized. Examples of diseases in which nonsense mutations
arc known to be among the causes include: cystic fibrosis (cystic fibrosis
transmembrane conductance regulator gcnc.h Duchenne muscular dystrophy
(dystrophin), beta thalassacmia (p-globin);
► insertion or deletion occurs when the addition or removal of a base alters the
reading frame of the gene. It may be small (one or a few base pairs within a
gene) or large (an entire gene, several genes, or a large section of a chromosome).
This change affects every codon beyond the point of mutation and thus may
dramatically change amino acid sequence. A Ira mesh i ft mutation may occur
when a number of inserted/ddeted nucleotides in a DNA sequence is not in.
multiples of three nucleotides. Frameshift mutations can lead to a premature
end to translation of the mRNA or the formation of an extended polypeptide.
The resulting protein is usually nonfunctional. Frameshift mutations arc known
to be a factor in Crohn's disease, familial hypercholesterolemia, cystic fibrosis,
colorectal cancer as well as other cancers;
► duplication is when extra, copies of a chromosomal region arc formed, resulting in
different copy numbers of genes within that area of the chromosome. This type
of mutation may alter the function of the resulting protein;
► inversion is a chromosome rearrangement in which a segment of a chromosome
is reversed end to end. An inversion occurs when a single chromosome undergoes
breakage and rearrangement within itself. An inversion docs not involve a loss of
genetic information, but simply rearranges the linear gene sequence;
3J 0. The Effects of Mutations 173

> translocations when segments of two chromosomes arc exchanged (may interrupt
gene sequences):
► loss of heterozygosity is a loss of one allele of a genetic locus. Il is a common
genetic event in cancer.
Table 3.1 Z The types of mutations

Sitent Amino acid sequence is not changed

Missense Amino acid sequence is changed
Nonsense Amino acid sequence is changed to a stop codon, therefore truncating
the protein

Insertion Addition of one or more extra nucleotides

Deletion Deletion of one or more extra nucleotides

Duplication Multiple copies ol chromosomal region

Translocation Interchange of regions from different chromosomes
Reverse! of the orientation ol chromosomal region
Inversion Loss of one allele
Loss of heterozygosity

Fig. 3.43. The scheme illusiraTing the different types of mutations: duplication, deletion. inversion and
translocation of DNA. A region of a chromosome before and after mutation is indicated

Sequence Divergence and Molecular Clock. The Relationship

between Mutations and Polymorphisms
Mutations arc changes in the genetic sequence, and they arc a main cause of
diversity among organisms, i.c., the mutation js one of the fundamental forces of
evolution.How do mutations afreet population'?
Most changes in protein sequences occur by small mutations that accumulate
slowly with time. Point mutations and small insertions and deletions occur by chance,
probably with more or less equal probability in all regions of the genome, except for
174 Chapter 3. Sene expression and protein synthesis

hotspots al which mutations occur much more frequently. Most mutations that change
the amino acid sequence arc deleterious and will be eliminated by natural selection.
Few mutations arc advantageous; those that spread through the population eventually
replace the former sequence. When a new variant replaces the older version of the
gene, it is said to have become fixed in the population.
Some genes or protein sequences may accumulate mutations at a relatively constant
rate (c.g., I change per million years). If this rate of change is reliable, it is possible
to calculate the time of divergence according to the number of differences. E.g., if a.
gene, which mutates al a rate of I bp per 100,000 years has 6 bp different, divergence
occurred 600,000 years ago. This concept is called the molecular clock. The molecular
clock provides a valuable means of estimating evolutionary timescales from genetic
and biochemical data.
The sequences of genes or proteins can be compared among species and used to
build phylogenetic trees. Closely related species typically have few sequence differences,
while less related species tend to have more. For example, cytochrome C is a small
hemeprotein and it. is associated with the inner membrane of the mitochondrion. Il
is an essential component of the electron transport chain (see Ch. 4). The amino acid
sequence of cytochrome C has been analyzed in over 100 species. Fig. 3.44 shows
the sequence comparisons for cytochrome C between human and cither rat or yeast.
Alignment of cytochrome C sequence of human and rat has a high, level of homology
(top panel), whereas alignment of cytochrome C sequence of human and yeast has
a lower level of homology (bottom panel). Sequence similarity searches can identify
<■ ho motogou s* protoi n s or go nos by dcicc Li ng excess si m i la ri ty — statist ical ly sig n i lica n t
similarity that reflects common ancestry. Substitutions within the primary structure
of the protein are relatively constant, over lime and. therefore, cytochrome C can be
considered as a potentially useful molecular clock. The results shown in Fig. 3.44,
indicate that the evolutionary path leading to the human species diverged with that of
yeast far before the divergence from a rat.
Betow: Alignment of human and rat cyloc hrome c amino acid sequences using BLAST.
Sequence similarity between the two proteins is 01%

Human Cyl C: MGDvEkGKKIFjMkCSCChTvEkGGKHKTGPNLHGlFGRkTGQAFGySyTAANKNKGIW 61

Alignment MGDvEkGKKIFi- KCt QChT'/EkGGKHKTGPNLHGlFGRkTGOa G+SyT ANKNKGl W
HatCytC: MGDvEKGKKlFvOKCAQCHTvEKGGKH^TGPNLHGLFGFiKTGQAAGYSYTDANKNKGlTW 61

Human CytC: GEDTlMEYLEnPkkyiFGTkMiFvGikkkEERADUAYIKKATnE 105

Alignment GEE'TD^EYlEnPkkyiPGTkMiF GlKKK ERADliaylkkaTNE
Rat Cyt C: GEDT LME YLEnFkk Yl PGTkMiFaGikkkGERaDu AYLKKATnE 105

Below: Alignment of Human and yeast cytochrome c amino acid sequences using BLAST.
Sequence similarity between me two proteins is 64%

HumanCylC: GDvEKGKKlFlMKCSCX;HTvEKGGKHKTGPNLHGLFGRKTGQlAPGYSYTAANKNKGllwG 61
Alignment G 1-KG +F -tC QChTvEkGGHK GFNLHG-i-FGR -t-GQA GySyT AM K +1-W
Yeasl Cyt C: GS^KKGATlFkTRClQCHTVEkGGFHKVGPNLIHGiFGRhSGQAEGySyTDANIKKMVLVVD 66

Human Cyl C: EDTlMEYLEnPKKYIPGTkMiFvGIKKKEERaDLIAYLKKA 102

Alignment E+ + EYL NPKKYIPGTKM F G+KK+-Hfi DLI YLKKA
Yeasl Cyl C: ennmseyltnfkkyipgtkmafgglkkekdrndutyijkka 107

Fig. 3.44. Alignment of cytochrome C from various sources

3.10; The Effects of Mutations 175

Interestingly, traits can be gained and lost multiple times over the evolutionary
history of a species.
The frequency of new mutations in a single gene or organism over time is not
limited to a single type of mutation. The rate at which mutations accumulate is
a characteristic of each protein. A protein evolves by mutations, followed by either
elimination or fixation. The presence of two allelic variants in the population, is
called polymorphism. Importantly, mutations by themselves do not classify as
polymorphisms. Polymorphism is a DNA sequence variation that is common in the
population. In polymorphisms, there arc two or more equally acceptable alternatives
and to be classified as a polymorphism, the least common allele must have a frequency
of I % or more in the population. If the percentage of occurrence is smaller, a random
occurrence — mutation, takes place. Polymorphisms arc responsible for many of
the normal differences between people, eg., blood type (Ch. 7.9). Ah hough many
polymorphisms have no negative effects on a person’s health, some of these variations
may influence the risk of developing certain disorders.
Single nucleotide polymorphisms (SNPsJ are the most common type of genetic
variation among people. Each SNP represents a difference in a single DNA nucleotide.
For example, some DNA molecules in the same population may have a T-A base pair
al a particular nucleotide siter whereas other DNA molecules in the same population
may have a C-G base pair at the same site. This difference constitutes an SNP. The
SN P defines two alleles for which there could be three genotypes among individuals in
the population: homozygous chromosomes or heterozygous chromosomes with T-A
in one chromosome and C-G in the homologous chromosome.
SNPs may occur within coding sequences of genes, non-coding regions of genes,
or in the intcrgenic regions (regions between genes). SNPs within a coding sequence
do not necessarily change the amino acid sequence of the protein, due to degeneracy
of the genetic code. Importantly, SNPs that arc not within a coding region may affect
gene expression. About 3 million SNPs that are relatively common in the human
population have been identified, of which about I million arc typically used in a search
for SNPs that might be associated with complex diseases. Each DNA polymorphism
serves as a genetic marker for its own location in the chromosome. SNPs responsible
for a disease can occur in any region that can ultimately affect the expression,
structure, and activity of the protein. Examples arc SNPs in transcription factor
binding domains, in promoter regions, in areas involved in transcript processing, such
as SNPs al intron-exon boundaries, which may cause defective splicing, or in mRNA
processing signal sequences such as polyadenylation signal regions.
Most SNPs have no effect on health or development. However, in some cases SN Rs arc
very important in the study of human health, e.g., response to certain drugs, susceptibility
to environmental factors such as toxins, and risk of developing particular diseases. SNPs
arc often used to track the inheritance of disease genes within families. For example, the
major disease gene for breast cancer in women is the gene BRCA I.l. For women who cany

' Vertebrate genes and proteins have names (typically strings of words) and symbols, which
are short identifiers (typically 3 to S characters). For example, breast cancer type I. susceptibility
protein is a protein that in humans is encoded by the BRCA I gene. BRCA I is a human tumor
suppressor gene that is responsible forrepairing DNA The tyrosinase — TYRgenc is responsible
for the production of the enzyme tyrosinase, which is the key enzyme in the formation of
melanin pigment. These symbols are usually, but not always, coined by contraction oracronymic
176 Chapter 3. Sene expression and protein synthesis

a mutant allele of B RCA I, the lifetime risk for breast cancer is higher than for women who
arc not carriers. The importance of the genetic risk factor can be expressed quantitatively
as a relative risk, which equals the risk for disease in the individuals who carry the risk
factor as compared with the risk in those who do not. Another example is cytochrome-P
450. Cytochrome-P 450 (CYP) enzymes play a role in the synthesis of many molecules,
including steroid hormones, certain lipids and bile acids. Additionally, CYP enzymes
arc essential for the metabolism of many medications, and internal substances, such as
toxins. CYP enzymes account for 70-80% of enzymes involved in drug metabolism.
Polymorphisms in cytochrome P450 genes can affect the function of the enzymes. The
effects of polymorphisms arc most prominently seen in the breakdown of medications.
Depending on the gene and the polymorphism, drugscan be metabolized quickly or slowly.
If a cytochrome P450 enzyme metabolizes a drug slowly, the drug slays active longer and
less is needed to get the desired effect. A drug that is quickly metabolized is broken down
sooner and a higher dose might be needed to be effective. Therefore, polymorphisms in
CYP can lead to different drug responses or loxicitics.
Hereditary diseases arc diseases or disorders that arc inherited genetically.
Hereditary diseases arc passed on from one generation to another through defective
genes. The chromosomes in the humans arc responsible for passing the traits from the
parent to the offspring.
The most common hereditary diseases include Huntington’s disease, myotonic
dystrophy, neurofibromatosis, cystic fibrosis, Duchenne’s muscular dystrophy,
haemophilia, Marfan syndrome, a and p-thalassemia, fragile X syndrome, sickle cell
disease, etc. The following example illustrates how gend s defects causes disorders.
Albinism is one of the inherited disorders. Affected individuals typically have very-
fair skin and white or light-colored hair. Long-term sun exposure greatly increases the
risk of skin damage and skin cancers. All forms of albinism also cause problems writh
the development and function of the eyes.
A defect in one of several genes that produce or distribute melanin causes albinism
(Table 3.I3). As can be seen from the Table 3.I2, there are more than 300 different
mutations in 777? gene that arc associated with the disease. Il encodes a protein
tyrosinase of 529 amino acids. 7TJ? gene is located on chromosome llql4.3 and
consists of five exons (Fig. 3.45). The defect may result in the absence of melanin
production, or a reduced amount of melanin production. The defective gene passes
down from both parents to the child and leads to albinism.
Humans arc diploid organisms because they have two alleles at each genetic
locus, with one allele inherited from each parent. If the two alleles arc the same,
they arc homozygotes. If they are di Ik rent, they arc hcterozygoies. In the case of
hctcrozygotes, the individual can express cither one or a combination of the two
traits. Oculocutaneous albinism (OCA) is inherited in an autosomal recessive manner,
meaning that both copies of a gene in each cell have mutations. Most often, the
parents of an individual with an autosomal recessive condition each carry one copy of
the mutated gene, but they do not show signs and symptoms of the condition. Ocular
albinism (OA) is much less common and inherited in an X-linkcd pattern meaning
that the mutated gene that causes the disorder is located on the X chromosome, one

abbreviation of the name. .All human gene names and symbols can be searched online al the
«HGNC database of human gene names — HUGO Gene Nomenclature Committee*.
3J 0. The Effects of Mutations 177

of the two sex chromosomes.In males, one altered copy of (he gene is sufficient to
cause OA, because males have only one X chromosome.
Table 3.13. Mutations detected lor albinism associated genes*

-i
□CAI TYR 11q14-q21 303
□CA2 □CA2 15q11.2-q12 154
□CA3 TYRP1 9p23 16
OCA4 SLC45A2 5p13.3 78
□CAS ND 4q24 1
□CAO SLC24A5 15q21.1 2
□CA7 C1OORF11 1Oq22.2-q22.3 1
OA1 GPR143 Xp22.3 114
LYST CHS1 1q42_1-q42.2 53
HPS1 HPS2 1Qq23.1-q23.3 31
AP3B1 HPS2 5q14.1 20
HPS3 HPS3 3q24 7
HPS4 HPS4 22cen-q12.3 13
HPS5 HPS5 11p14 11
HPS6 HPS6 10q24.32 9
HPS7 DTNBP1 6p22.3 2
HPSfi BLOC1S3 19q13.32 2
HPS9 BLOC1S6 15421.1 1

OCA. oculocutaneous albinism. Tn® OCA1A is Hue most severa type with a complete lack of melanin picductic-n truoughout
life, while 1Fie milder forms OCAlB, OCA2, OCA3, and OCA4 slutw soma pigrront accumulation ever time. OA, ocular albinism
is a genelic confttiwi that primarily affects the eyas, OA reduces the coloring (pigmentation) of the iris, and the retina, which
is the ligm-sensifert tissue in the hack of the ey®. Il is clwacterizeo by severely impaired sharpness of vision and problems
with combining vision from Doth eyas to perceive depth (stereoscopic vision). OA Hoes not significantly affacl ths color of
the skin and hair. LYST, lysosomal 'trafficking regulator. HPS. Hemiansky-Pudlak syndrome, is a genetically heterogeneous
group of autosomal recessive disorders characterized principally by oculocutaneous afflinism (OCA), blooding tendency, and
progressive pulmonary fibrosis. AP3B1. adaptor related protein complex 3 subunit beta! TYRPi. tyrosinase-related protan 1.
is an enzyme in the melanin biosynthesis pathway, catalyzing She oxidation of 5.6-dinydroxyindblo-2-carbbxylic acid (DMCA)
monomers into melanin (see Ch. 7.).
1 Source: human gene mutation database. 05 Doc, 2013; JMD: not determined.
mjZOiSw AJTp2-yS GyS'JMFnc GrESl-bt Gly47GyS
wuzeTm AigE-2Th Gtfs:DCTfp GrrXiAig -EtyEiArg AM+'.&Sfli ASp+44Gly
amK&M:- Aip77Qn -E^^Aig GkiZtSLyS &y97ftg JVQJtf’Gifl ALp+4fiAST
waffifiTni AiCsTTGly ■
GyflS+V GkJ-l'JTwfTTi GiyJTvoi AJg«Htjty GmfiMs
jwg,nGTaffl wgTTTrp
j t Gy5SAfg GuZ2iLys leiBZ-As; Arg+CPLii rsrrr.
A-J2 i2LjS ASfCVnr GyiEE’yi Gki2EZ'Rm Hfifhug — ijSECk AipjfijAi- AflMCZ'Hffn Qu-32flMa
A^IiITnf
*jg2 ilTnr A^jiKTyr Gj-mEZ'^
A-zp i35Tyr ByMfiZyi GariSTtanm nsiOGri AnSSEPra ■SiiYKlKh.- wwtBSw GmSlKh
AJp2lTE*fl ALf i-HlAa- GyfiSE.7yf -ZlyluEdVC i«302Ajp am33iTw ATO4O3E&- ZVu.WhMJ
Aiy2l713y ‘ ‘ - --
■SpsEflAig - lOCMroi
ijh —• bfcsKKjn amS’JiCk ija'ETain- Jij-g^ZEiDu GiL*Z(lA!4< AM^EiGk.
A/p2r.rTi-p ASf2*jGiy GysSiEor HasSujug A£fl37l~nr BnSTSLyt aam2K«i GkM lET-witi AiMiOGtr
.AK|2?Kih A&p^SASn Gyt'llTyf ClySSG*. »tc2&£T-,r Air3.' i Tye GtrtiraTwiB AjjiHTip EkMrjWj-g Grfiat’&in
*jp233Trp ASjMiiEi^ GrSEToffn Giy4i>wg Ki23"ni ALfTjffiL-jiS G.yjJoGkj Anj+S«K GkMStArg Fn»6DS«

c
az; " Zj<.: ^lEaZLy: Qy3461affnAg-43&A5|l GAMAc-E^llTriMo rafffi

Excm 1

Bfl22F'rY Pdc iE+CjS Sari53TDffli TrpZ3£S« TyriETcmi

LOuE+QTam FnoiTEi; EariaSTrf TrpcraS-Tomi TyrtETam
I C-Ar34&jty FioE-iEaic
GkMEaq Sa-2?3Frc
1
Exon 3

G4ji34€-¥3
■jlrHZPJVU.
EoSECfj.
EnrSEiAnj
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HMlMASfl $a42*R-B
tw+C4Frc' Eom*2Fitc
lju2iEUct — ■"......... - TipflSSTom
■EjfWA/n tyau GIlu'ET tfiil
vu^— Sw33H3Jy HK3B3Tjf EnOSOPra -.Du^Pvai Ti-jJ+HK^
LAd)PJO 5tr4+Gjy _rp272Aj-g varTi'Fna ldu2BEFtid ~^-—
TwEEAia •irtSEJ'wg WQM.'HLyS Ti-psOOia.
L|Sl3H3b E^mEjZT<mi7 T.ytfTKyz - —
vit™"
SinF—
th LGu2®Eei- TjfSTCys rts3STTjr PnMSboj Tjf4l i»-h-
Ly5142w» E^ECTflffl TijtlflAX Lbj3'»2W9 voCTSPtk HfSXlAIfl WJGT7AM FTO+CfcLCu Tjrt/BCyt
LySMiMCI Efl'TillLdu TifMC/s MeE.'k "kQBlBuiu MaCTDK WM3K1FTW FlIM l2AM
lydJ+ETrr Ear75Pra TipliTiilTi Mafl32Tnr WsCTOTw FlD+lTbM
Ly533TTV nwiffiSw TipBDwg. RltiMtihj FTC-4'J I LUj
UaiTDUu TfpBZTwm PiwEsThr V3ME7Q^
MGEiffivai Ty1M9Cy£ SuMSwg M3m2.'Ftk
Wnr.Tw Ty f IBlCfS Bk3Sasi
HM1V3 TyfEGE+lj'

Fig. 345. Database of tyrosinase (Tr/7) gene mutations. The mutations identified in 0CA1 for each
exon are indicated. Nomenclature for tne description of mutations: Arg52Lys — Arginin al position 52
is reptacedi by tysine
178 Chapter 3. Sene expression and protein synthesis

Sickle cell disease is one oft he most common inherited blood disorders. The disease
is caused by a single point mutation of the p-globin gene (WBB), on chromosome
II. As a result, a sixth amino acid in. the chain is valine rather than glutamic acid
(Fig. 3.46). The disease is inherited in an autosomal recessive manner, which means
both copies of the gene in each cell have mutations, i.e., a baby born with sickle cell
disease inherits a gene for the disorder from both parents. When both parents have
the genetic defect, there is a 25% chance that each child will be born with sickle cell
disease. If a child inherits only one copy of the defective gone (from either parent),
there is a 50% chance that the child will carry* the sickle cell trait.
Wild-type haemoglobin DNA Mutant haemoglobin Dna

Normal haemoglobin Sickle cell haomoglotiin

4-------- 4 ‘-QF
Fig. 3.46. Missense mutation and HDS. The 20A >T mutation in me HOB gene results in the production
of haemoglobin S (HBS)

Different genetic mutations in the p-globin gene (HBB) on. chromosome

II produce different variants of abnormal hemoglobin (S, C or E):
► IIbS (sickle hemoglobin) — The 20A-*T mutation (A al position 20 is replaced
by T) in HBB gene results in the production of he moglobin S (HbS) (Fig. 3.49);
► I IbC (hemoglobin C disease) — The 19G-*A mutation (G at position 19 is replaced
by A) in the HBB gene results in rhe production of hemoglobin C (HbC):
► HbE (hemoglobin E disease) — The 79G->A mutation (G at position 79 is
replaced by A) in the H B B gene results in the production of hemoglobin E (HbE).
Abnormal hemoglobin is stiff, sticky, and distorts the red blood cell into a sickled
shape (sec Ch. 1.2). Individuals affected with sickle cell disease have at least one copy
of the HbS variant of the hemoglobin P-globin subunit. In the most severe form of
sickle cell disease, also known as sickle cell anemia, two copies of HbS are inherited. 1 n
other types of sickle cell disease, one copy of HbS and a copy of a different abnormal
p-globin subunit arc inherited. A common type of SCO is hemoglobin SC disease,
where one copy of HbS and HbC each arc inherited.
Another X-1 inked recessive condition is red-green color blindness (i.e., a red-green
color blind allele). Affected individuals have difficulties in distinguishing between some
shades of red, yellow, and green. Red-green color blindness is caused by mutaiiori s of
different genes located on different chromosomes.
3.11. Recomninanl ONA Technology 179

Haemophilia A is an X-linked bleeding disorder when the blood cannot clot

properly due to a deficiency of a cloning factor called Factor VIII. Different point
mutations, insenions, small and large deletions, inversions arc associated with the
disease.
Cystic flbrosis is a disorder that results in. a thick, sticky buildup of mucus in the
lungs, pancreas, and other organs. The disease is caused by the mutation of a single
gene, the cystic fibrosis transmembrane regulator gene, which is located on the long
arm (q) of chromosome 7 (position 31.2).
Some forms of breast cancer can be hereditary. Two genes arc associated with
hereditary breast cancer, BRCL4/ and BRCL42, which arc tumor suppressor genes.
The BRCzt/ gene is located on chrl7q, and the BRC42 gene is located on chr!3q.
Carriers of mutated SRC4/ c7?id BRC.42 genes are at an increased risk for both breast
and ovarian cancers.
Maple syrup urine disease is an inherited disorder characterized by a deficiency
of certain enzymes required to metabolize the three branched-chain amino acids,
leucine, isolcucinc and valine, in the body properly. Different types of mutations can
cause the disease.
Some hereditary diseases resulting from duplications or deletions of chromosomal
regions can be seen in Table 3.14.

Table 3.14. Examples of diseases resulting from duplications or deletions of chromosomal regions

The d
Charcot-Marie-Tooth disease type il Duplication 17p12

Hereditary neuropathy witii pressure palsies Delation 17p12

Smith-Magen is* syndrome Deletion 17p11.2

Williams*b-Beuron syndrome Deletion 7q11_23

" in a small numbar of cases, people win Srmth-Maganfis syndrome tave irtnei itod tne deletion or mutation from an
unattncled molder who Harf me genetic cltartge only in her egg ceils. This phenomenon is called germline mosaicism.
” in a small percemage of cases, people with Williams syndrome inherit the chromo&omal deletion from a parent with the
condition.

3.11 RECOMBINANT ONA TECHNOLOGY

Recombinant DNA technology is widely used in the clinical diagnosis, in prenatal
screening lor genetic diseases, recombinant protein production, such as insulin and
growth hormone, as well in gene therapy, vaccine production, etc.
Recombinant DNA technology relates to the usage of three main, tools:
> enzymes: restriction enzymes, polymerases, ligases;
> vector: is a section of DNA that can incorporate another DNA fragment without
losing the capacity for self-replication. If the fragment of DNA includes one or
more genes the process is referred to as gene cloning;
> host organism: the host cell copies the cloned DNA using its own replication
mechanisms. A variety of cell types arc used as hosts, including bacteria, yeast
cells, insect cells, mammalian, cells.
180 Chapter 3. Sene expression and protein synthesis

Recombinant DNA technology includes:

► Cleavage of DNA. e.gM the gene or fragment of interest, by restriction
enzymes;
► DNA ligation that allows to join together DNA molecules from different
sources:
► Nucleic acid hybridization to identify any specific sequence of either DNA or
RNA based on the unique ability of two complementary nucleic acid strands to
interact with each other;
► DNA cloning, amplification of DNA fragment by polymerase chain reaction
(PCR) using thermostable polymerase., insertion of the desired recombinant
DNA into the vector, subsequent transformation ofcci Is/organ isms and obtaining
the product of the recombinant gene mostly in the form of the manufactured
protein;
* DNA sequencing allows to determine the sequence of nucleotide bases in DNA.
* Reverse transcription PCR (RT-PCR) allows using RNA as a template. RT-
PCR: RNA is first transcribed into complementary DNA (cDNA) by reverse
transcriptase enzyme from total RNA or mRNA. The cDNA is then used as the
template;
► Quantitative PCR (qPCR) and RT-qPCR allow to detect, characterize and
quantify nucleic acids for numerous applications. qP'CR and RT-qPCR arc
widely used in. diagnosis of in fee tious disease (c.g., human hepatitis viruses), SNP
genotyping and allelic discrimination, somatic mutation analysis, chromatin
immunoprecipitation quantification, gene expression analysis. RNAi validation,
microarray validation, pathogen detection. In contrast to conventional PCR, the
accumulation of amplification product is measured as the reaction progresses, in
real time, with product quantification after tach cycle.

Recombinant DNA
Recombinant DNA (or rDNA) is made by combining DNA from two or more
sources (Fig. 3.47). In practice, the process often involves combining the DNA of
different organisms. Recombinant DNA is an artificially made DNA strand that may
or may not occur naturally, but is engineered specifically for a purpose to be used in one
of the many applications of recombinant DNA.
Restriction enzyme is an endonuclease that recognizes a short, specific sequence
in DNA and cleaves DNA al these sites along the molecule. These regions arc called
recognition sequences and arc randomly distributed throughout the DNA. They arc
produced by bacteria in order to defend against bacterial viruses. Restriction enzymes
can be isolated from bacterial cells and used in the laboratory to manipulate fragments
of DNA. e.g., to cut DNA into smaller fragments. The cuts arc always made al specific
nucleotide sequences. Different restriction enzymes recognize and cut different DNA
sequences. The names of restriction enzymes arc derived from the genus, species, and
strain of the bacteria that produce them; for example, the enzyme EcoRI is produced
by E cofi strain RY 13 and specific recognition sequence is GAATTC, Bam HI is
produced bv strain II and specific recognition sequence is
GGATCC (Fig. 3.48), etc.
3.11. Recomninanl ONA Technology 181

Fig. 3.47. RecoinOinanl DNA technology, simplified scheme. Steps include: cutting the desired ONA
and vector Dy restriction enzymes, gene amplification Dy PCR; inserting tne gene into the vector,
transferring tne vectors into host organism, and obtaining the products of recombinant gene
182 Chapter 3. Sene expression and protein synthesis

L
5'-ATGGATCCAA-3' Bam H1 5-ATG-3r 5?-GATCCAA-3?
3’-TACCTaGGTT-5’ —“ 3-TADCTAG45' 3?-GTT-5’
I
5-GAATTC-3’ Eco R1 5-G-37 5-AATTC-3’
3'-CTTAAG-5: ----------- 3'-CTTAA-5' 3-G-5’
I

Fig. 3.48. Schematic representation of restriction enzymes Bam anti fcnffl cleavages. SamHI
recognizes the DMA sequence 5r-GGATCC-3r, and introduces a single-strand cut between tne G and G
nucleotides. This recognition site is a palindrome: the apposite strand also reads 5'-GGATCG-3’ and will
be cut in the same manner. EroRl recognizes tne DMA sequence 5'-GAATTC-3', and introduces a singte-
strand cut between the G and A nucleotides. Tnis recognition site is a palindrome: tne opposite strand
also reads 5'-GAATTG-3r and will be cut in the same manner. The cleavage sites of EccRl is shown

Gel Electrophoresis
Gel electrophoresis is a technique commonly used in laboratories to separate
charged molecules like DMA, RNA, and proteins according to their size (Fig. 349).
Samples

Marker ABC

1_200bp

I.ODObp
909 bp
BOO bp

700bp

BOO bp

SOObp

400bp

390 bp

200 bp

tOO bp

Fig. 3.49. Gel electrophoresis of DMA (left) and proteins (right). The drawing showing DMA hands
separated on a gel (left panel). The length of the DNA fragments (A, B, C) is compared to a marker
containing fragments of known length, illustration showing protein bands on a gel rrignt panel). The
size of me protein is compared to a marker containing fragments of known length
3.11. Recomninant ONA Tec neology 183

Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is a technique that makes many copies of
a certain segment of DNA m w?ro. Starting with a single DNA molecule, PCR can
generate 100 billion identical molecules in just a few hours. A great advantage of PCR
is that it can copy one specific segment from within a tremendous length of DNA. The
technique is so precise and powerful that its starling material docs noteven have to be
purified DNA.
Basic PCR ingredients include:
* DNA template to be copied:
► primers, short stretches of DNA that initiate the PCR reaction, designed to bind
to either side of the DNA that has to be amplified:
► DNA nucleotide bases (dNTPs) that arc needed to make a new strand of DNA;
► thermostable polymerase, e g., Jatj, P/w, Phujicn;
► buffer to ensure the right conditions for the reaction.
PCR is performed in thermal cycler (PCR machine). The PCR machine increases
and decreases the temperature of the sample in automatic, programmed steps
(Fig. 3.50):
► Initially, the mixture is heated to denature, or separate, the double-stranded
DNA template into single strands;
► the mixture is th.cn cooled so that the primers anneal to the DNA template;
► DNA polymerase begins to synthesize new strands of DNA, starting from the
primers.

Double-stranded
DNA

Fig. 3.50. Three main stages of PCR incEude denaturation. annealing, ana elongation

At the end of the first cycle, each double-stranded DNA molecule consists of one
new and one old DNA strand. PCR then continues with additional cycles that repeat
the aforementioned steps. The newly synthesized DNA segments serve as tern plates
194 Chapter 3. Sene expression and protein synthesis

in subsequent cycles, which allow the I)NA of interest to be exponentially amplified

millions of times.
After PCR has been completed* the data arc analyzed by c.g., agarose or
polyacrylamide gel electrophoresis, sequencing or restriction, digestions (discussed
below), etc.
PCR is used in gene cloning and manipulation, gene mutagenesis, construction of
DNA-based phylogenies, diagnosis and monitoring of hereditary diseases, amplification
of ancient D NA, analysis ofgenetic fingerprints for DNA profiling, detection of pathogens
in nucleic acid tests for the diagnosis of infectious diseases, etc. Pig. 3.51 provides an
example of molecular diagnosis of herpes simplex virus in clinical samples by PCR.

Fig. 3.51. PCR analysis. PCR products from assay controls and 12 clinical specimens. Samples in lanes
1,2, 4, and S are negative for the test viruses (internal control [iC] product visible oniy|. Samples in
lanes 9 and 10 are HSV**-1 positive, in lanes 11 and 12 are HSV-2 positive, and in lanes 6 and 7 are
CMV positive, and me sample in Eane 5 is VZV positive. Molecular mass (MW) markers (with selected
sizes in base pairs) are shown at both ends of the gel; neg — negative control (nuctease-free water).
‘ from Julian Druce et al. J. Clin. Microbiol. 2002; 40:1728-1732. * * — HSV — Herpes Simplex
Virus, CMV—Cytomegalovirus, VZV — Van cel la-zoster virus

DNA Ligation
DNA ligase is a specific type of enzyme that facilitates the joining of DNA strands
together by catalyzing the formation of a phosphodiester bond.
► If two DNA molecules have matching ends, they can be joined by DNA ligase:
► Restriction enzymes and DN.A ligase are often, used to insert genes and other
pieces of DNA into plasmids during DNA cloning.

Restriction Fragment-Length Polymorphisms (RFLP)

RFLP is a technique that exploits variations in homologous DNA sequences. Il
is a difference in homologous DNA sequences that can be delected by the presence
of fragments of different lengths after digestion of the DNA samples of interest with
specific restriction endonucleases.
► In RFLP analysis, the DNA sample is broken into pieces (and digested) by
restriction enzymes;
► The resulting restriction fragments arc separated according to their lengths by gel
electrophoresis.
The RFLP probes arc frequently used in genome mapping in genotyping, paternity
tests, hereditary disease diagnostics, etc.
3.11. Recombinant DNA Tecnnnlogy 185

The niost wc 11 - know n cxa mplc is t he R F L P d tic Lo (1-globi n gc nc mu talion (Fig. 3.^ 2).

Mst II Mst II Mst II

| [Tgtobin gene | |
Normal DMA
CCTGAGG><
CCTGAGG CCTGAGG

1150 base pairs 200

base pairs

Mst Ji Mst II
I Gene with sickle cell mutation |
Mutant ON A
CCTGAGG CCTGAGG

1350 base pairs

Fig. 3.52. RFLP resulting from p-Qlohin gene mulatiDiL in tie normal ce I, the sequence corresjpndino
to 5th to 7in am no acids of the p-gio&in peptide is CCTGAiGGAG mat is recognized by Msm Digestion
wiitn MsELI generates fragments 0.2 kb and 1.2 kb (upper panel), in tbe sickle cells there is A >T
mutation (discussed above), and the site is not recognized Dy Maty

Northern, Southern and Western Blotting

Northern, Southern, and Western arc three blotting techniques used to delect a
specific RNA. DNA or protein molecule in. a sample (Fig. 3.53). During blotting, the
macromolecules arc transferred onto a membrane from the gel and made to bind with
a specific nucleic acid or antibody that aid in the detection.
► Northern blotting is used for detecting RNA fragments:
► Southern blot is used for detection of a specific DNA sequence in DNA samples;
► Western blotting is used to detect a particular protein in a mixture. The probe
used is neither DNA. not RNA, but antibodies. The technique is also called
dmniunobloLtingfc.
106 Chapter 3. Sene expression and protein synthesis

Cell with DNA. RNA & Protein

(RNA) |
Southern Blot
(Restriction enzyme)

=-
Northern Blot
(Single stranded)
Western Blot
(Deanatured polypeptides)

XI I
«
E
□
a.

Ell *

I
□
-i—1

I I
u

a
a
e Agarose Acrylamide
0.5—50 Kbp 0.5-10 Kb 10-120 Kd

Transfer separated samples to membrane

ft Single stranded Single stranded Primary antibody

e
complementary complementary to specific polypeptide
S
□ JO DNA or RNA to DNA or RNA to Use Secondary antibody
0-
ka E
specific sequence
(restriction fragment}
specific sequence
(transcript)
to detect/amplify primary
E

Detect labeled probe on membrane

I
□
ft
ft
CC Sample contains specific Sample contains specific Sample contains specific
DNA restriction fragment RNA transcript Polypeptide
(e.g., mRNA)
Can measure fragment Can measure polypeptide
size and amount Can measure fragment size and amount
(single vs. repeated) size and amount (level of expression)
(level of expression)

Fig. 3.53. Comparison of Southern. Northern, and western Plots

3.11. Recomninanl ONA Tecnnology 187

Questions and situational problems

LA particular gene codes for a mature mRNA containing 900 bases, which is
translated into a 40 k Da protein. A mu Lam form of the gene created by a single
point mutation yields an 830-base mature mRNA yielding a 37 kDa protein with
modified enzymatic activity. The analysis shows that the mutation has resulted
in a 22 amino acid deletion within the protein. What is the most likely effect of
the mutation? Explain.
2. Agarose get electrophoresis of PCR products from samples taken from a
healthy, a carrier, and an affected individual revealed DNA bands of different
sizes. Please indicate what sample correspondingly belongs to healthy, carrier,
and affected individuals, if you know that a shortcrabnormal DMA fragment is
obtained as a consequence of nonsense mutation.

1000 bp—
750 bp—*■

400 bp—

3. A missense mutation results in the presence of a different amino acid than was
encoded by the parental sequence. This type of mutation can have a drastic effect
or no effect at all depending on the importance of the amino acid and the type
of amino acid that replaces it. Some amino acids are structurally similar and
may be able to act as viable substitutes for each other. For example, changing
one acidic amino acid to another may not affect the final protein, buL changing
a polar amino acid to a nonpolar amino acid will likely disrupt the structure.
Please explain what mutations occur in //BB gene of abnormal hemoglobin and
their effect on the function of the protein.
5. The 7YB gene encoding for tyrosinase from two individuals was analyzed. It
has been found that one individual has a wild type TK/f and the other one has
the mutation. On the gel electrophoresis, the control unaffected and the mutant
have no difference in the mobility, i.e., no change in size. What type of mutation
had most likely occurred?
6. The starting sequence of a gene changed from jAUGTTCGACGTGp to
jAUGTTTTCGACGTG^. What type of mutation is this? To answer the
question, please explain:
1) what the mutation is?
2) what types of mutation do you know?
3) what arc the possible outcomes of mutations?
7. Scientistsstudying the genetics ofa congenital disease analyzed the chromosomes
of the patient and found a large portion of chromosome twenty-th rec in
188 Chapter 3. Sene expression and protein synthesis

chromosome one, and a small pan of chromosome one i n chromosome twenty-

three. What type of mutation is this? To answer the question, please explain:
I ) what the mutation is?
2) what types of mutation do you know?
8. A single nucleotide polymorphism changes one nucleotide in a gene sequence.
The gene 300 bp size was analyzed for mutations. As a result, bUAA|Sicodon was
found. What type of mutation is this? To answer the question, please explain :
1) what is a single nucleotide polymorphism?
2) how it affects the protein function?
9. Which of the following mutations would be least detrimental to the function of
a protein and why?
1) Silent:
2) Frameshift;
3) Deletion of two nucleotides:
4) Nonsense;
5) Misscnse.
10. Samples were tested for sickle cell disease by PCR. The results were analyzed
by agarose gel electrophoresis. Lancs 1, 3, 5, 7, and 9 arc PCR-amplified, but
undigested DNA (364 bp), and lanes 2,4,6,8, and 10 are Dde [-digested DNA.
Lancs I £2 arc from a known carrier DNA (PA/fh 4 bands). Please, identify the
genotype in lanes 3£4, S&b, 7&8g 9& 10.

3456709 10

364 bp—
291 bp—
201 bp—

90 bp—

pA/ps

11. What is the Zac opcron? What happens in terms of the Zac opcron when lactose
is absent from the growth medium?
12. You are working with a piece of DNA of the sequence:

5' -TATTG AGCTCC CCGG AT-3'

3-ATAACrCGAGGGGCCTA-5'

You cut the above piece of DNA with a restriction enzyme that recognizes the
sequence 5’GAGCTC and cuts on. the 31 side of the A within this sequence.
Please, draw all products that you get after digestion. Label all 51 and 3’ ends.
13. Once transcription is complete, gene expression is controlled by...?
14. Why denaturation stage is important in PCR? To answer the question, please
explain:
3.11. Recomninanl ONA Technology 189

I.) what is the basic principle of PCR?

2) what arc the components of PCR?
3) what arc the main stages of PCR?
I5. The deficiency of glucose-6-phosphate dehydrogenase (G6PD) is one of rhe
most common inborn errors of metabolism worldwide. Th is congenital disorder
generally results from mutations that arc spread throughout the entire gene
of G6PD. Three mutations for G6PD, 362AAAAGAGGG^AAACAUGGG,
^CACAAUCAC-CACCAUCAC. kAUGACUAAA-+AUGGCUAAA arc
responsible for severe decrease of the protein activity. What types of mutations
arc these? To answer the question please:
1) explain what the mutation is:
2) describe the types of mutation you know;
3) explain the difference between germ l ine and somatic mutation?
 Chapter 4
STRUCTUREANDFUNCTIONS
OF BIOLOGICAL MEMBRANES.
CELL SIGNALING PATHWAYS

4.1. General Description of Membranes: Composition and Structure

42. Transmembrane Transport
4.1Transmembrane Signal Transduction
4.4. Signal Transduction Through Intracellular Receptors

41. GENERAL DESCRIPTION OF MEMBRANES: COMPOSITION

AND STRUCTURE
Membranes embraces all cells and cell organelles, playing an. important role in
maintaining their structure and functions. All membranes have general structural
characteristics. However, the plasma membrane, as well as membranes of the
endoplasmic reticulum, Golgi apparatus, mitochondria and the nuclei have prominent
compositional specificity; they all have unique components and, therefore, unique
functions.
Main functions of membranes are:
► separation of a cell or organelle from the environment., creating the specific inner
content;
* regulation of transport into the cells and organelles and back;
► determination of the specificity of Lhe intercellular contacts;
► perception of the signals like hormones or other signaling molecules from the
extracellular environment.
The coordinated functioning of membrane systems, including receptors, enzymes,
transporters ensures the cell homeostasis and quick response to environmental changes
by intracellular metabolism regulation.
Biological membranes consist of lipids and proteins, connected via n on-covalent
bonds. Lipids form lipid bilayer and protein molecules embedded into it (Fig. 4.1).
Lipid bilayer is formed by two layers of amphiphilic molecules, mainly phospholipids
and cholesterol, which interact with each, other by their hydrophobic parts, forming
inner hydrophobic layer of membrane, and hydrophilic groups — apolar heads* —
turned outwards and contact with water medium.
Lipids of the membranes. Three main types of lipids form the membrane;
glyccrophospholipids. glycolipids, and cholesterol (Ch. 7.L Table 7.1). The most
commonly found are glyceropbospholipids. derivatives of phosphatidic acid (Ch. 7.9,
Fig. 7.31). The main glyccrophospholipid in most of the membrane types is
phosphatidylcholine (Fig. 4.2).
4.1. General Description of Membranes: Composition and Structure 191

PhospMpids
imerior

Fig. 11. The cross-section through me plasma membrane

Membrane phospholipids contain saturated unsaturated, including

polyunsaturated fatty acids. The unsaturated fatty acids arc more prevalent in the
membrane phospholipids, therefore the hydrophobic layer of a membrane is Uli id
at body temperature, which governs the possibility for the conformational lability
of membrane proteins.
Sphingolipids — derivatives of amino alcohol sphingosine. Sphingosine can be
converted into ceramide by acylation — the process, when the fatty acid is bound to
the NH-,-group of sphingosine. The fatty acid chain residue delineates the type of
ceramide. Also, some polar groups can be bounded to OH-group of the ceramide.
Depends on such apolar head* structure, there arc different types of ceramides:
sphingophospholipids and glycolipids. The structure of a polar group of the single
sphingophospholipid sphingomyelin is similar to the phosphatidylcholine. The
myelin sheath of the neuronal axons contains a lot of sphingomyelins. Glycolipids
arc carbohydrate derivatives of ceramide: a sugar is attached to the ceramide
lipid backbone. Depending on. the type of this sugar — there arc cerebrosides and
gangliosides (Ch. 7.9. Fig. 7.35). Glycolipids arc located in the cell membrane with
the carbohydrate groups extruded from the cell surface (Fig. 4.1). These carbohydrate
groups serve as cell recognition factorsand some of them act as the antigens.
Cholesterol is a compound of the membranes of all animal cells: it restricts
membrane fluidity and is responsible for membrane stiffness. The cholesterol
molecule positioned in the hydrophobic zone, parallel to the ^hydrophobic tails*
of phospho- and glycolipids. The hydroxyl group of cholesterol, as the *hydrophilic
heads* of phospho- and glycolipids, turned into water medium. The molar ratio of
the cholesterol and other membrane lipids is 0.3—0.9. Cytoplasm membrane has the
highest value of this ratio.
192 Cnapief 4. Structure and functions of biological membranes. Cell signaling pathways

Polar (riydropfMfic) Head

Choline------

N+(CH3)3
Phosphate——CHZ

0
Glycerol -—m 0=P—O’

CHn—CH-CHP

6 o

Fig.4.2. Role of glycerophosplwlipid phosphatidylcholine and cholesterol in the formation ol membrane

lipid bilayer. Left — phosphatidytcholine. Saturated radicals ol the fatty acids positioned close to each
other in ine pnospnoiipid molecules and this results in me formation of hydrophobic bonds between
mem; the double bond in the radical causes me formation of a bend, distance between hydrophobic
radicals of fatty acids increases and these bonds are much weaker. Membrane phospholipids contain
a lot of unsaturated tatty acids, therefore the hydrophobic layer is fluid at me body temperature, which
ensures the conformational [ability of me membrane proteins. Cholesterol molecule consists of four-
ring rigid hydrophobic structure and flexible hydrocarbon chain. An -OH group of me Ihird carbon
atom of cholesterol molecule plays role of a -polar head*. Cholesterol is a factor, that restricts the
fluid state of inner membrane layer

Increased cholesterol concentration in the membranes restricts the phospholipid

fatty acid chains mobility, which in turn affects the conformational lability of
membrane proteins and decreases the possibility of their lateral diffusion. There is
a diversity in the lipid content of membranes, and, apparently, the presence of one
or another lipid is determined by the verity of these molecules functions in. the
membranes.
4.1. General Description of Membranes: Composition and Structure 193

Main functions of membrane lipids are:

► formation of the lipid bilaycr — the structural backbone of the membranes;
► prevention of free movement of molecules and ions through the membrane,
which ensures the specific content of intracellular fluid and the fluid inside
organelles;
► providing a favorable environment for the membrane proteins functioning;
► serving as an ^anchor* for the surface proteins:
► panicipation in the signal transduction, coming to the cell as hormones or other
signaling molecules (primary1 messengers).
Disruption of the lipid bilaycr can have a detrimental effect on general membrane
functioning and the cell itself.
Membrane proteins. Membrane proteins differ depending on their position in the
membrane (Fig. 4.3).

Fig. 4.3. Membrane proteins: 1,2 — integral itransmembrane) proteins; 3,4,5,6 — surface proteins.
Part of the polypeptide chain of tne integral proteins is submerged into lipid bi layer. Those protein parts,
wtiicn interacts wim hydrocarbon chains of fatty acids, mainly consist of non-polar amino acids. Protein
parts, located in the polar «neads* area, nave lots of hydrophilic amino acid residues. Surface proteins
have different ways to attach to the membrane: 3 — connected to the integral proteins; 4 — bonded to
me polar - heads* of lipid Drlayer; 5 — ^anchored- in the membrane with me help of short hydrophobic
end domain; 6 — '’anchored* in the membrane with the help of covaienlty bonded acyl residues

Membrane proteins, which arc in contact with the hydrophobic pan of the lipid
bilaycr must be amphiphilic, holding a non-polar domain. The hydrophobic property
of this non-polar domain is guaranteed by the following;
► the amino acids residues contacting the lipid bilaycr mainly arc hydrophobic;
* a lol of membrane proteins arc covalently bound to the fatty acids residues
(acylated) that make them more hydrophobic.
Acyl residues bound to proteins provide their ^anchoring* to the membrane and
the possibility of the lateral dillusion. Along with that, membrane proteins undergo
posit ran slat ional modifications, such as glycosylation or phosphorylation. The
glycoproteins serve as receptors for compounds such as hormones, as cell attachment
and as cell — cell recognition sites. Viruses and bacteria also bind to these sites.
Membrane proteins take part in:
► selective transport of metabolites into the cell and back;
» hormonal signaling;
► formation of ^coated pits* which takes part in cndocytosis and cxocytosis;
► immune response:
► catalytic reactions:
► intercellular interactions, which provide the formation of tissues and organs.
194 Chaplet 4. Structure and functions of biological membranes. Cell signaling pathways

Oilier and inner layers of the same membrane have different lipid and
protein composition. This membrane structural specificity was characterized as
transmembrane asymmetry.

4.2. TRANSMEMBRANE TRANSPORT

The main function of the membranes —is the transport of the molecules, complexes
of organic molecules and ions in and out of the cell, retention of the substances
necessary to the cell and clearance of the unnecessary ones. Transport of ions and
organic molecules through the membranes can go down the concentration gradient —
passive transport, and against the concentration gradient — active transport.
Passive transport hits the following routes (Pig. 4.4):
► simple diffusion without transporter proteins. This way cell gels O3, NH... H3O,
C()3, urea, hydrophobic low molecular weight substances, for example, steroid
and thyroid hormones (Pig. 4.4, I );
► facilitated diffusion with the help of transporter proteins — for example, tissue
glucose transport with the helpofGLUT4 transporters (Fig. 4.4, 2);
► passive symport — transport of two types of ions down the gradient concentration
in the same direction, for example. H and HPO42' (Fig. 4.4, 3);
► passive anti port — transport of ions down the gradient concentration in opposite
directions, for example, HC03 and CT (Fig. 4.4,4);
► ions diffusion by the protein channels — for example. IT, Ca2 Na’,. K'. Most
of the channels arc regulated by the specific ligands and by the change of the
transmembrane potential (Fig. 4.5).
Passive transport
Membrane Membrane
outer inner
surface surface

Fig. 4.4. Transmembrane transport by The gradient concentration

4.2. Transmembrane Transport 195

Fig. 4.5. Ca3* channel of trie endoplasmic reticulum, regulated by the inositol 1,4,5-hisphDsphate
(IP*). IP3 (inositol 1 AS-trispnosphate! is formed during nydrolyzis of membrane lipid PlP?
•fpnospnatiOylinositol 4,5-bispnosphate) Dy the enzyme phospholipase C. IP* binds to the specific sites
of Ca7, channel protomers in the ER membrane. Protein conformation changes and channel opens —
Caf • goes into cell cytosol by the gradient concentration

Active transport. The primary-active transport happens against the gradient

concentration and uses ATP energy with transmembrane adenosine 5’ triphosphatases
(ATPases), for example, Na\ K‘-ATPase. Il ’-ATPaso, Ca2'-ATPase (Pig. 4.6). FL­
AT Base acts as proton pump, creating an acidic pII in the cell lysosomes. Ca2! -AT Base
in the plasma membrane and membrane of the cytoplasmic reticulum maintains the
low Ca* concentration in the cytosol and formsan inner cellular depo of Ca‘ in. the
mitochondria and endoplasmic reticulum.
A secondary-active transport is happening due to gradient concentration of one of
the transporting compounds (Fig. 4.7), which is commonly formed by the Na+/K+-
ATPasc. consuming the ATP.
If the active site of a transporter protein binds the compound with higher
concentration, this changes its con formation and increases the affinity to the substance,
which pass through the cell against the concentration gradient. A secondary-active
transport can be of two types — symport (transport of two types of the substances in
the same direction), and antiport (transport in opposite directions).
Membrane transport of nano particles and macromolecules — endocytosis and
exocytosis. Endocytosis provides transport of macromolecules, ex. proteins, nucleic
acids, polysaccharides and bigger cargo molecules from the extracellular space inside
the cell. Binding of such macromolecules happens at specific membrane locations —
coated pits. Endocytosis, which is happening with the help of specific receptors,
embedded into such coaled pits, helps cells consume specific substances, so it is called
a receptor-depend ent endocytosis.
Macromolecules, for example, peptide hormones, enzymes, intracellular matrix
proteins, lipoprotein complexes, arc secreted into the blood or intracellular space
by means of exocytosis. This process allows to excrete substances from the cell,
accumulated in the secretory granules. Mainly, exocytosis is regulated by the change
in. the calcium ions concentration in cell cytoplasm.
196 Cnapief 4. Structure and functions of biological membranes. Cell signaling pathways

Membrane Membrane
outer inner
surface surface
Ca2+ — ATPase of the plasma
and endoplasmic reticulum
membranes helps to maintain
the low concentration of calcium
in the cell cytosol and also
calcium intracellular depot is
formed in the mitochondria
and endoplasmic reticulum.
Binding of two calcium ions In
Ca2*-ATPase sites, turned into
the cytosol, changes the charge
and conformation of the Ca2+-ATPase
conformation. The affinity
of the enzyme to ATP increases
and auto-phosphorylation is activated.
Binding the phosphate (P)
accompanied with the conformational
changes, Ga^-ATPase then closes
at inner side of the membrane
and opens from the outer side.
The affinity of the binding sites
to Ca2* ions decreases and they
dissociate from the enzyme.
Auto-de-phosphorylation is
activated by the Mg2+ ions (-).
Ca2+-ATPase lost its phosphate
and the affinity to the Mg2+ ions.
The enzyme conformation changes
and Ca^-ATPase gets back to its
initial state.

Fig. 4.6. Functioning of the C a2-ATPase

Membrane Membrane
outer inner
surface surface
Active symport - simultaneous
transfer of two substances in one
direction, one of which moves
against the concentration gradient
at the expense of the other moving
by the gradient concentration,
for example, Na*- dependent ( )
glucose transport into the gut ( )
Active antiport - transfer in
the opposite directions, one
of the substances moves against
the gradient concentration owing
to the other moving by the gradient
concentration, for example, Na41/ i
dependent Ca2+ transporter
in the saliva cells( )

Fig. 47. Secondary active transport

4.3. TransmemDrarte Signal Transduction 197

43. TRANSMEMBRANE SIGNAL TRANSDUCTION

A key feature of membranes — is the ability to perceive and pass inside the cell
signals from the environment. A signaling molecule, specifically interacting with a
cell receptor, called a primary messenger. A primary messenger could be a hormone,
neurotransmitter, eicosanoid, NO, growth factor or a physical factor, like a light
quantum. Hormones arc the molecules produced by specialized cells and secreted into
blood in response to the alteration of any specific parameter of the internal medium of
the body or external medium .
There are two main ways of signal transmission into the cell. Hormones and other
signaling molecules, which cannot pass through the membrane because they arc
hydrophilic or are too large, interact with receptors located on the plasma membrane
of targeted cell. Target cell is determined by its ability to bind selectively the signaling
molecule (primary messenger) with the receptor. Most of these receptors, binding a
primary messenger, activate the intracellular pathway, which results in the formation
of the second messengers. Second messengers pass the information further to the cell,
changing the velocity of metabolic processes.
A special group ofsignaling molecules — is hormones, which can pass through the
cell membrane due to their hydrophobic nature. Steroid and thyroid hormones belong
to this group. These hormones interact with receptors located in the cell cytosol or
even in the nucleus.
Hormones, interacting with the cell membrane receptors, transmits the information
to the system of proteins and enzymes, which, form a cascade of reactions, called a
signal transduction cascade or pathway . This cascade results in signal enhancement
of several hundred times. The time-response of the cell could be several minutes and
can be, for example, an activalion/dcaclivation of metabolic processes, a muscle
contraction, or content discharging from target cells.
There arc several types of membrane receptors:
► receptors, containing a subunit, which binds a primary messenger and has the
ion channel;
► receptors having a capacity for catalytic activity (insulin receptor):
► receptors which trigger with the help of G-proteins the formation of second
(intracellular) messengers, transducing a signal to specific cytosol proteins
and enzymes (Fig. 4.8). The examples of hormones acting through this type of
receptors arc glucagon and epinephrine.
Second messengers arc formed in the cell in response to the interaction between the
primary messenger (for example, a hormone) and a membrane receptor. Having small
molecule weight and diffusing through the cytosol quite quickly, they deactivated
specific proteins and then quickly deactivate or arc excreted from the cytosol.
The main second messengers arc:
► cAMP (cyclic 3’,5’ adenosine monophosphate);
► cGMP (cyclicguanosine monophosphate);
► IP1 (inositol 1,4,5-trisphosph.atc);
► DAG (1,2-diacylglyccrol);
► Ca+\
198 Cnapief 4. Structure and functions of biological membranes. Cell signaling pathways

© o o> @
Fig 4.8a Types of membrane receptors. Membrane receptors can De divided into tfiree groups:
Receptors: 1 — containing the subunit that binds a signal molecule and ionic channel, ex. acetylcnotine
receptor on synaptic membrane; 2 — snowing catalytic activity after binding a signal molecule, ex.
insulin receptor; 3,4 — transducing a signal to the adenylyl cyclase (AC; enzyme or phospholipase
0 (PLC) with me help of membrane G-proieins, ex. different epinephrine and acetylcholine receptors
and other primary messenger receptors

Physiologically important difference between a membrane and intracellular

hormone receptors — is the velocity of response: in the first case, it will be quick and
short, in the second — it's a long-lasting effect.

G protein-coupled receptors (GPCRs)

When hormone interacts with G protein-coupled receptor it thereby activates
phosphatidylinositol 4?,5’ — bisphosphatc signaling pathway or adcnylyl cyclase
signaling system.
Adenylyi cyclase signaling system consists of the following components (Fig. 4.9).
► Integral proteins of plasma membrane:
* R. — a primary messenger’s receptor, an activator of adcnylyl cyclase system;
* R, — a primary messenger's receptor, an inhibitor of adcnylyl cyclase system;
* Adcnylyl cyclase.
► Anchoring proteins:
* Ge— GTP/GDP-binding protein, having a^y-subunits, where the as-subunit
is an activating subunit;
* G — GT P/G DP-binding protein, having u.Py-subunits, where the a.-subunit
is an inhibiting subunit.
► Cytosolic enzyme protein kinase A.

Adenylyl cyclase signal transduction pathway

The primary messengers, activating the adcnylyl cyclase system, can be. for
example, hormones epinephrine and glucagon.
The receptor has a binding site for such primary messenger (hormone) at the outer
surface of the membrane and for the G protein (oJJy-GDP) at the cytosol surface of
the membrane. When a hormone (i.c., an activator of the adcnylyl cyclase system)
interacts with, a receptor (R), it changes receptor conformation. As a consequence,
the affinity of the receptor to G-protein is increasing. A.*hormone-receptor* complex
4.3. TransmemDrarte Signal Transduction 199

binds to G -GDP. which lowers the affinity of G - protein o-subunit to GDP, in

parallel enhancing the affinity to the GTP. As a result* GTP substitutes GDP in the
as-subunit binding site. This leads to conformational changes in the as-subunii and
hence, decreases its affinity to fty-subunits. As a consequence of these transformations,
a newly formed at-GTP-subunit starts a lateral move in the membrane towards
enzyme adenylyl cyclase (AC) (Fig. 4.9).

Active
PKA

E-OH ATp ADP E-O-POt

(non -ptrosphorytaied
(phDspticjrylaled
enzyme)
enzyme}

Fig. 4.9. Adeiiylyl cyclase pailiway. PKA — protein kinase A, R — regulatory subunit, C— cataliyic
subunit

Binding of the as-GTP to the regulatory site of adcnylyl cyclase changes the
conformation of the enzyme and turns it to the active state, which leads to increased
formation of the second messenger — 3 \5’-cyclic adenosine monophosphate (cAM P)
from ATP:
200 Cnapief 4. Structure and functions of biological membranes. Cell signaling pathways

As a result, the concentration of cAMP is increased in the cell. Molecules of

cAM P can reversibly bind to the regulatory subunits of protein kinase A (PKA). which,
has two regulatory subunits (R) and two catalytic (C) — R,Cr The R?C2 complex
itself has no enzymatic activity. As soon as cAMP binds to the regulatory subunits,
they undergo conformational changes and lose complementarity to C-subun its. and
complex R2C7 dissociates as a consequence. Catalytic subunits (C) thereby gain the
enzymatic activity.
Activated protein kinase A phosphorylates the hydroxyl groups of serine or
threonine in different cytosolic enzymes. The donor of phosphate group for this
reaction is ATP. The phosphorylation of the protein lowers or enhances its activity,
therefore changing the intensity of the associated metabolic processes.
In the case of the ft ( receptor inhibiting adcnylyl cyclase system) activation by
the signaling molecule, the G. is stimulated. The mechanism of GL functioning has
the same mechanism, as for the Gt protein, but with the difference, that binding
a. GTP to adcnylyl cyclase suppresses the enzyme activity. As a result the activity
of the whole system decreases.

Inactivation of adenylyl cyclase system

When the signal, triggered by binding a primary messenger with its receptor
is successfully transmitted, the adcnylyl cyclase system gets back to its initial
inactive slate and is ready to accept the next signal. This process of inactivation
has several steps.
Being in at-GTP complex and bonded to adcnylyl cyclase, us subunit acquires the
enzymatic (GTP-phosphatase) activity: it hydrolyzes GTP to GDP and P Formed
GDP molecule remains in the active site ofas subunit, changes its conformation and
weakens the affinity to adcnylyl cyclase. as-GDP complex dissociates from adcnylyl
cyclase, and as-GDP binds back to py subunits of Gs protein forming inactive Gs
complex. This cis-GDP dissociation from adcnylyl cyclase inactivates the enzyme, so
the cAMP synthesis terminates.
Phosphodiesterase — an ^anchored* enzyme of the cytoplasmic membrane, it
hydrolyzes the recently formed cAM P molecules down to AM P:
4.3. TransmemDrarte Signal Transduction 2(h

Asa result of this reaction, the cA MP concent ration in the cell drops consequently.
This triggers cleavage of the cAMP4R3 complex and increases the affinity of R- and
C-subunits, and inactive form of FKA (R3C3) thereby is formed.
One of the hormones, acting through the adcnylyl cyclase system. — epinephrine,
increases the heart rate, via stimulation of the cAMP rise in the cardiomyocytes.
Natural phosphodiesterase inhibitor — calle inc (coffee), decreases the rate of cAMP
cleavage, so its concentration rises in the cell. Therefore, taking high amount of
caffeine causes symptoms similar to the epinephrine action. There is list of drugs,
cardio stimulants, which arc inhibitors of phosphodiesterase.
Phosphoprotein phosphatase turns phosphorylated proteins and enzymes into their
dcphosphorylatcd form, so their conformation is changing, as the rate of the processes
they are involved in. As a result, the system regains the initial state and is ready to get
activated when the hormone would bind the receptor again. Thus, the balance between
the hormone blood concentration and the intensity of the target cells response is well
regulated.

Regulation of gene expression through adenylyl cyclase system

Some hormones acting through adcnylyl cyclase system not only regulate the rate
of metabolic pathways though phosphorylation of the key enzymes already present
in the cell bin can increase their synthesis by regulation of the genes expression
(Fig. 4.10). Activated protein kinase A enters the nucleus and phosphorylates the
transcription factor CREB (cAMP response element-binding protein). CREB is
constitutively bound to the DNA response element CRE (cAMP response element)
and is activated by phosphorylation. Activation of this transcription factor increases
the rate of transcription of the gene and thus the synthesis of the key enzymes or
proteins. The increase in their quantity leads to the elevation of the metabolic
pathway rate.
202 Chaplet 4. Structure and functions of biological membranes. Cell signaling pathways

mRNA
i
Specific proteins
I
Changes in uKiabolism
in target cells

Fig.4.10a Regulation of me genes expression by adenylyl cyclase system. FKA — protein kinase
A, R-regulatory subunits of FKA, C — catalytic subunits of FKA, ORES — CAMP response element
binding protein, CRE — CAMP response element, P — phosphate, AC — adenylyl cyclase

Phosphatidylinositol A^’-bisphosphate signaling pathway

Phosphatidylinositol 4\5’-bisphosphatc, also designated as PIP; pathway includes
the following components (Fig. 4.11):
► receptor (R) for a primary messenger of Pl P2 pathway;
► phospholipase C (PLC) — enzyme (surface protein):
► Gp|(. — G-protein consists of api(.jJY-subunits, where the a subunit binds to
GDP in inactive state and to GTP in activated state. Gm.is • anchored protein-in
plasma membrane:
► IPr DAG, Ca2* — second messengers.
4.3. TransmemDrarte Signal Transduction 203

Hwimorw

Calmodulin

CalmoduSin

Calmodulin n
Ca7--calmodulin
-la pendent
PK inactive

Calmodulin

Ca7-calmodulin
dependent
F'K aclive

t-OH 4J®

ATP ADP

Fig. 4.11. P hosphatixty linosilol T^’-bisphosphate (PFPJ signaling pathway

This signaling system includes the following proteins: calmodulin, protein kinase
C, Ca3' -depen de nt protein kinases, regulated Ca3' channels ofendoplasmic reticulum
(ER) membrane, Ca2 -ATPasesof the cell and mitochondrial membrane.
The binding of the activator (i.c.T its primary messenger) to the receptor (R) of
phosphatidylinositol 4\53 — bisphosphate (PIP.) signaling pathway initiates the
conformational changes in the receptor ( Fig. 4.1I). Therefore- the receptor affinity
to GW(-protein elevates. Then, binding of the complex primary messenger-receptor
to the Gp|f-GDP diminishesapif-subunit affinity to GDP along with the increasing
204 Cnapief 4. Structure and functions of biological membranes. Cell signaling pathways

affinity to GTP. Asa consequence, GDP is substituted for GTP in the binding site of
aPI< -subunit. This is followed by conformational changes in ap|(.-subunit and loss of
its affinity to py subunits, so the G?ir-proiein dissociation is taking place. The aJltc-
GTP subunit laterally drifts through the membrane to the enzyme phospholipase C.
Interaction of a^.-GTP with the binding site of phospholipase C changes the
conformation and activity of the enzyme, and the elevation of hydrolyzis rate of
specific membrane phospholipid — phosphatidylinositol 4,5-bisphosphate (P1P3) —
is taking place (Fig. 4.12).

(IP3)

Fig. 411 Hydrolyzis of phosphatidylinositol -4.5-Disphosphate (PlPJ

The output of this process — is formation of two products, so-called second

messengers of hormone signal: diacylglyccrol {DAG), which remains in the membrane,
taking part in the activation of protein kinase C. and inositol 1.4,5-trisphosphatc
<1P}, which is, being hydrophilic, makes its way out to the cytosol. Thus, we have
here a bifurcation of the signal, initially accepted by the receptor. IP. binds to the
specific sites of the ER membrane Ca2’-channel, triggering conformational changes
and Ca2' -channel opening. As a consequence, Ca2' leaks to the cytosol by the gradient
concentration, as calcium concentration in ER is 3—4 times higher than in cytosol.
Important, the channel is closed when the IP. is not present in cytosol.
Calmodulin, a small protein, is present in the cytosol of all cells; it has 4 binding
sites forCa2 . As concentration rises, calcium binds calmodulin very actively, forming
complex |4Ca2'-calmodulin |. This complex interacts with calmodulin-de pendent
protein kinases and other enzymes and potentiates its activity. Activated Cae­
cal moduli n-dc pendent protein kinase phosphorylates proteins and enzymes, which
then become activated and their metabolic activity escalates loo.
Increased cytosol Ca2' concentration potentiates the Ca2 interaction with inactive
protein kinase C (PKC). Binding Ca2’ to PKC stimulates its drifting in the plasma
membrane and allows to interact with membrane phospha tidylser inc (PS) negatively-
4.3. TransmemDrarte Signal Transduction 205

charged «heads». DAG, sitting al the specific sites of protein kinase C, potentiates
its affinity to Ca2 even more. Eventually, the active state of PKC is formed al the
membrane inner surface: |PKC-Caj4-PS-DAG|. Now, being in that complex, it can
phosphorylate specific enzymes.
The PIPn system has a short-term activity, and right after the cell response the
phospholipase C, protein kinase C and Ca3'-calmodulin-dependent enzymes arc
getting inactivated. The aP[f-subunit in complex with GTP and phospholipase C
shows enzymatic (GTP-phosphatase) activity and hydrolyzes GTP to GDP. Being
bonded to GDP. ct?1(,-subun it loses its affinity to phospholipase C and gets back to its
inactive state, incorporating again into the complexafiy-GDP (Gprj£-protein).
Dissociation of aJlfC-GDP from PLC inactivates the enzyme, so the hydrolyzis of
PIP..terminates. Increased cytosol Ca2' concentration activates Ca2'-ATPascs in the
ER and plasma membranes, which pumps out calcium from the cytosol. The Na7
Ca2 - and H'/Ca2 -transporters arc also taking pan in this process, working by the
active antipon mechanism. Since Ca2’ concentration is decreasing, the dissociation
and inactivation of Ca2 '-calmodulin -depen de nt enzymes is taking place, as well as the
loss of the PKC affinity to the membrane I ipids and its activity loo.
The IP, and DAG, formed during the system activation, can interact again and
turn into the PIP-,.
Phosphorylated proteins and enzymes arc being transformed into their
dcphosphorylatcd state by the phosphoprotein phosphatase, which changes its
conformation and activity; thus, the whole signaling system returns to its initial state.

Receptors with catalytic activity

Catalytic receptors arc enzymes. They can be activated by hormones (insulin),
growth factors, cytokines. When activated, these rcccptors-cnzymcs phosphorylate
specific protein by their —OH groups of tyrosine residues, that is why they arc called
tyrosine protein kinases (Fig. 4.13). The signal, accepted! by catalytic receptor, can be
transduced to the nucleus with the help of specific mechanism, where it suppresses or
potentiates gene expression.
Insulin receptor — is an example ofa catalytic receptor and contains four subun its —
two a and two p. Whereas a-subun its located at the outer surface ofcell membrane, the
p-su bunks stretch across the membrane bi layer. The insulin binding site is formed by
N-terminated domains of a-subun its. Receptor catalytic site is located on intracellular
domains of p-subun its. The cytosol part of the receptor holds several tyrosine residues,
which can be phosphorylated and dcphosphorylatcd.
As insulin attaches to the binding site of a-subunils, the cooperative
conformational changes arc taking place in the receptor. In parallel, P-subun its
reveal a tyrosine kinase activity and catalyzes trans-auto-phosphorylation of
several tyrosine residues: one subunit phosphorylates the other and vice versa.
Such phosphorylation leads to a change of the charge, conformation and substrate
specificity of the tyrosine-protein kinase (Tyr-PK). Tyr-PK phosphorylates specific
cellular proteins, which, therefore, got the name insulin receptor substrates a (IRS).
These proteins, in their turn, play important role in the activation of the following
proteins and enzymes:
206 Cnaptef 4. Structure and functions of biological membranes. Cell signaling pathways

Fig. 411 Activation of the insulin receptor. Phosphodiesterase transforms cAMP into AMP and cGMP
into GMP. Fnospnoprotein phospnaiasedephosphoryiates specific phosphorylated proteins. GLUT4-
giucose transporter in me glucose-dependen t tissues (adipose and skeletal muscles). Tyrosino-protein
phosphatase depnosphoryiates p-subunits of the insulin receptor
4.3. TransmemDrarte Signal Transduction 207

► phosphoprotein phosphatase (PPP) which dephosphorylatcs specific

phosphoprotcins:
► phosphodiesterase converting cAM P to AM P and cG M P to GM P;
► GLUT 4 — glucose transporter in the insulin-dependent tissues, therefore
glucose uptake by the muscle and adipose cells increases;
► tyrosine protein phosphatase — the enzyme dephosphoryJating (J-subunits of
insulin receptor;
► nucleus regulatory prole in s/tnmscription factors — proteins regulating expression
of the genes Tor specific enzymes.

Signal transduction by primary messengers (growth factors)

with Ras- and Raf-proteins
The implementation of the effects of other signaling molecules — growth factors —
happens also with the help of catalytic receptors, which contains one polypeptide
chain, but forms dimers after primary messenger binding. All receptors of this type
have an. outer glycosylated domain, transmembrane (a-helix), and intracellular
domain, which shows protein kinase activity when activated.
The dimerization triggers their catalytic intracellular domains activation, which
then starts a trans-auto-phosphorylation of the serine, tyrosine and threonine amino
acid residues. Binding the phosphate groups leads to the formation of binding sites for
specific cytosol proteins on the receptor and triggers activation of the protein kinase
pathway of signal transduction (Fig. 4.14).
Binding of one molecule of the growth factor (GF) to the binding site of the
receptor results in the receptor (R) dimerization and trans-auto-phosphorylation. A
phosphorylated receptor gains an affinity to Grb2-protein (specific adaptor protein
that hinds to phosphotyrosinc residues on growth factor receptors). A newly formed
complex [GF-R-Grb2] interacts with cytosol protein SOS. The change in SOS
conformation makes them able to bind the anchoring membrane protein Ras-GDP.
The function of the complex [GF-R-Grb2-SOS-Ras-GDP] is to lower the Ras-
protcin affinity to the GDP and increase the affinity to the GTP.
The substitution of GDP for GTP changes the conformation of Ras-protein,
which separates from the complex and interacts with Raf-protein near the membrane.
Complex [Ras—GTP-Raf] demonstrates protein kinase activity and phosphorylates
enzyme MEK-kinase {mitogen-activated protein kinase kinase). Activated MEK-
kinase phosphorylates MAP-kinasc by its threonine and tyrosine residues. Wien -
PO32' group binds to the MAP-kinasc amino acids radicals it changes its charge,
conformation and activity. The MAP-kinasc cascade terminates at a gene transcription
factor, thereby regulating transcription of certain genes involved in cell survival and
proliferation.
206 Cnapief 4. Structure and functions of biological membranes. Cell signaling pathways

OH
[MEK kinase]i ■ [ M E k kinase 1

7\—
ATP ADP

<e>
. MAP kinase ■ [ M AP kinase I
ATP^-J /

AC
ADP

Fig. 414 MAP-kinase pathway. Tne receptors of such kind are activated Dy epidermal growth factor
(EGF), nerve growth factor (NGF) and other growth factors. Grb2 — protein, interacting with growth
factor receptor (growth receptor binding protein); SOS (GEF) — GDP-GTP exchanging factor (Ras
subfamily of guanine nucleotide exchanging factors); Ras — G-protein (guanosine triphosphatase
family); Raf kinase — in its active state phosphorylates MEK-kinase; MEK kinase — MAP kinase
kinase 'mitogen-activated protein kinase kinase); WAP kinase - mitogen-activated protein kinase
4.3. TransmemDrarte Signal Transduction 209

Receptors will] guanylyl cyclase activity arc also catalytic receptors. Guanylyl cyclase
catalyzes the formation of cGMP from GTP, which is one of the most important
second messengers in the intracellular signal transduction (Fig. 4.15).

cyclase

E-OH

Inactive protein Active protein

kinase G kinase G

Fig. 4.15. M emu ran e bound guanylyl cyclase activity regulation. Membrane bound guanytyl cyclase —
transm em brane glycoprotein. Biding site of a signal moiecute is located in me external ligand-bounded
domain. The internal catalytic domain manifests tne catalytic activity in result of its activation

There arc two types of guanylyl cyclase receptors. One type is a mem brane­
spa lining receptor in the plasma membrane with the external binding site for the
primary messenger atrial natriuretic peptide (AN Pl, regulating fluid homeostasis in
the organism. The other type of guanylyl cyclase receptor exists in the cytoplasm and
is a receptor for nitric oxide (NO), which is a neurotransmitter (neurohormone). NO
is a lipophilic gas that is able to diffuse into the cell. Thus, this receptor is an exception
to the rule that intracellular receptors arc gene transcription factors.
As primary messenger binds to the receptor, the guanylyl cyclase is getting activated
and catalyzes the transformation GTP to cyclic guanosine-3 \5’-monophosphalc
(cGMP) — a secondary messenger. The concentration of the cGMP in the cell is
getting higher. Molecules of cGMP can. bind reversibly to the regulatory sites of
the protein kinase G (PKG), which consisted of two subunits. Four molecules of
cG M P change conformation of an activity of the enzyme. Now active PKG catalyzes
phosphorylation of certain cytosol proteins and enzymes. cGMP — elevating drugs or
drugs releasing NO (such as glycerol trinitrate I arc used to treat a variety of disorders,
such as angina pectoris because the final response for NO is relaxation of the arteries,
erectile dysfunction (drugs inhibiting cGM P phosphodiesterase), heart failure (using
synthetic natriuretic peptide).
210 Cnapief 4. Structure and functions of biological membranes. Cell signaling pathways

4A SIGNAL TRANSDUCTION BY INTRACELLULAR RECEPTORS

Hydrophobic hormones (by i is chemical nature) like steroid and thyroid hormones,
can diffuse through the membranes; therefore, their receptors arc located in the cytosol
or in. the nucleus of the cells.
Cytosol receptors arc connected to the chape rone-protein, which preserves a
premature activation of the receptor. Nuclear and cytosol receptors of steroid and
thyroid hormones have a DNA-binding domain, regulating the interaction between
hormone-receptor complex and DNA regulatory sequences and so influence the
transcription rate.

Cascade reactions regulating the transcription

First, a hormone passes through the cell plasma membrane. Then in the
cytosol the interaction of a hormone and a receptor is happening. The complex
hormone-receptor enters the nucleus and binds to the DNA regulatory sequence
of nucleotides — enhancer (Fig. 4.16) or silencer. The crucial point — is that the
accessibility of the promoter of a gene for RNA-polymerase is increasing when
complex hormone-receptor is bound to the enhancer, and it is decreasing when
the complex is bound to the silencer. The transcription rate of specific structural
genes can be elevated or suppressed, accordingly. Then the synthesized mRNAs
leave the nucleus and move to the cytosol of the cell where translation lakes place.
A rate of translation of certain proteins increases or decreases, in response. Lastly,
a number of proteins, regulating metabolism and functional activity of the cell is
changing.
Each cell holds the receptors, included in different signal transduction pathways,
which transform all environmental signals into intracellular. The number of receptors
for each type of primary’ messenger can vary from 500 to over 100 000 per cell. They
can be positioned in the membrane al a certain distance from each other, or just at the
specific loci.
4.4. Signal Transduciion Dy intracellular Receptors 211

Steroid Hormone

Cytosof
Shaperon

intracellular
receptor

Hormone binds Hormone Receptor

to receptor complex
in cytosol
Cytosol

Nucleus

Complex
Honrone-Receptor
binds to enhancer
and activates specific
genes transcription

mRNA

♦
Specific proteins

♦
Metabolic changes
in the targeted cells
y
Fig. 116. Hormonal signal transduction via intracellular receptors
212 Cnapief 4. Structure and functions of biological membranes. Cell signaling pathways

Review tests
L Choose the correct answer.
<rs-su burnt of the G-protein, bounded to GTP, activates:
A. Receptor.
B. Protein, kinase A.
C. Phosphodiesterase.
D. Adenylyl cyclase.
E. Protein, kinase C.
2. Match lhe figure with the letter.
Function:
A. Regulates the catalytic receptor activity.
B. Activates phospholipase C.
C. Transforms the protein kinase A into its active form.
D. increases Ca3' concentration in the cytosol.
E. Activates protein kinase C.
Secondary messenger:
1. cAMP.
2. Ca2'.
3. IP3.
3. Match the figure with the let ter.
Function:
A. Ability to laterally diffuse in the membrane bilayer.
B. Binds the enhancer in the complex with primary messenger.
C. Shows enzymatic activity when binds to primary messenger.
D. Can bind G-protein.
E. Interacts with phospholipase C during the signal transduction.
Receptor of:
1. Insulin.
2. Epinephrine.
3. Steroid hormone.
4. Choose the correct answers.
The conformational lability of the protein-transporters can be influenced by:
A. Cholesterol content in the membrane bi layer.
B. Membrane electric potential change.
C. Binding specific molecules.
D. The fatly acid composition of the lipid bilayer.
E. The amount of the transported substance.
5. Match lhe figure with the Idler.
A. Ca1' channels of the ER.
B. Ca2‘-ATPase.
C. GLUT 4.
D. N'a* dependent Ca2' transporter.
E. Na'/K* ATPasc.
1. Transport Na' by the gradient concentration.
2. Act l bro ugh the facilitated diffusion.
3. Transport Na' against the gradient concentration.
4.4. Signal Transduciion Dy intracellular Receptors 213

6. Choose tfee correct answer.

Peptide hormones interact with the receptors which arc:
A. 1 n the cell cytosol.
B. Integral, membranes proteins of Lhe targeted cells.
C. In the cell nucleus.
D. Covalently bonded to PIP,.
7. Choose the correct answer.
Binding the growth factor (GF) to the receptor ( R) leads to:
A. Complex GF—R localization change.
B. Dimerization and trans-auto-phosphorylation of the R.
C. R conformation change and binding to the Gs-prole in.
D. Complex GF— R movement.

Situational problems
1. Card io myocytes contractile rate increases as Ca2+ levels in cytosol rise.
Releasing of Ca2 to the cytosol from endoplasmic reticulum is regulated by
cAMP-dependant transporters in ER membranes. Along with that, the cAMP
concentration in cytosol is regulated by two signal molecules.— epinephrine and
acetylcholine. Epinephrine, binding to j},-receptors, elevates the concentration
of cAMP in the cardiomyocytes and stimulates the cardiac output, and
acetylcholine diminishes cAMP concentration and, therefore, depress
myocardial contractility. Explain why, using the same signal transduction
pathway, these two primary messengers cause the di Here nt cellular responses in
the heart muscle. For that:
a) draw the scheme of signal transduction for epinephrine and acetylcholine;
b) point out the di (Terences in the pathways for these two primary messengers;
c) explain the opposite biological effects of these signal molecules.
2. Researchers have discovered significant changes in. the gene of some protein,
which is a substrate for the insulin receptor. What impact on the insulin signal
transduction would such changes have? For the answer:
a) draw the scheme- of insulin signal transduction in the target cell:
b) name proteins and enzymes, activated by insulin in the target cells;
c) explain the functions of these enzymes.
J. Steroid hormone calcitriol activates an intake of dietary calcium, increasing
the amount of Ca.2' transport proteins in the gut cells. Explain the calcitriol
mechanisms of action. For that:
aidraw a general scheme of a signal transduction by steroid hormones and
explain the process;
b) name the molecules, which synthesis is activated by a hormone in the nucleus
of a target cell;
c) explain the function of these molecules in the cytoplasm of the target cell.
4. Copy Table 4.1 to your notebooks and 11 1 I i t i n.
214 C napter 4. Structure and functions of biological memo fanes. Cail sig nah ng pathways

Tatle 4.1. Adenylyl cyclase and phosptalidylinosiwI 4,,5’-Disphosphale (PlPJI signaling pathways

An example of the primary messenger

integral membrane protein, complementary binding the primary

messenger primary

Protein, activating the enzyme oi the signaling system

System enzyme, forming the secondary messenger(s)

Secondary messenger^) of the system

Cytosolic enzyme(s), interacting with the secondary messenger

Regulatory mechanisms (specificto the system) ot the metabolic

enzyme activity

Mechanisms of the secondary messenger s concentration

decrease in the targeted cell

The cause of the membrane enzyme activity decrease (specific lo

the system)

5. The sense of smell — is one of the possible ways to de Leet a signal from the
environment in animals, including humans. It is now acknowledged that human
can delect about 10000 types of smells (odors). Receptors, receiving the scent,
can define molecules differed by only one carbon atom or by one functional
group. The molecule of an odorous substance — odorant, binds to granule­
adsorbent in a mucus composite, covering the olfactory cells in the nasal
cavity. Complex odorant-adsorbent interacts with cell membrane receptor.
The final part of this signal transduction is the opening of cAMP-depcndcnt
Na -channels in the olfactory cell membrane. This results in excitation of the
olfactory neuron and transduction of action potential via olfactory nerve to the
central nervous system. Explain the specificity of all proteins and non-protein
molecules structure, which takes part in the odorant substance transduction.
For that:
a) copy Fig. 4.17 in your notebook and add the required compounds to the
scheme;
b) draw a general structure of the membrane receptor, which interacts with the
complex odorant-adsorbent, if known that such receptors are analogs to the
P^-cpincphrinc receptors;
c) name the protein, with one of the protomers is being able to move laterally
through the membrane and Lransducc the signal to the membrane enzyme;
describe its functions;
d) name the enzyme, which, in its active form increases the cAM P concentration
in the olfactory cell; write down the reaction that is catalyzed by this enzyme;
e) make a description of cAMP-depcndcnt Na' channels functioning:
4.4. Signal Transduciion Dy intracellular Receptors 215

f) explain, how docs the system get inactivated, after being activated by an
odorant and name the enzymes and proteins involved in this inactivation
process.

Adsorbent
granules
MuCuS

4 T ■*0 Q
•
> B1 _ Channel
0

Fig. 117. The scheme of an odorant molecule Din ding to an olfactory cell receptor
 Chapter 5
CATABOLISM AND CELLULAR
BIOENERGETICS

5.1. Energy Producing and Energy Utilizing Reactions. The ATP-AD P Cycle.
Specific and Common Catabolic Pathways
52. Common Pathway for the Final Oxidation of Metabolic Fuels
53. Tricarboxylic Acid cycle (TCA cycle)
5.4. Mitochondrial Electron Transport Chain and Oxidative Phosphorylation.
Thermogenesis
5.5. Oxygen Toxicity

5.1 ENERGY PRODUCING AND ENERGY UTILIZING REACTIONS. THE

ATP ADP CYCLE. SPECIFIC AND COMMON CATABOLIC PATHWAYS
Adenosine triphosphate (ATP) is an exclusive cellular energy currency. ATP
provides energy to drive many processes i n living cells, c.g , muscle contraction, nerve
impulse propagation, transport of molecules, ion gradients, and chemical synthesis.
ATP consists of adenine attached to a sugar (ribose), which in turn is attached to a
triphosphate group (Fig. 5.1). ATP molecule contains 2 high-energy bonds between
yand p, and a and |3 phosphate groups. These high-energy bonds store more energy
than ordinary chemical bonds. During the hydrolysis of y phosphate - 7.3 kcal/mol
energy is released.
Hjgh-irergy Iwnifc

0 0

HO

‘N

f Adenine ]

OR OR:
[ Ribosg

Fig. 5.1. Chemical structure of adenosine triphosphate (ATP)

5.1. Energy Producing and Energy Utilizing Reactions. The ATP-ADP Cycle. Specific... 217

The total quantity of ATP in the human body is about 0.2 moles, which is
approximately 100 g and is enough to support cellular demand only for few seconds.
Therefore, the cells need a con slant ATP supply. In adult humans, approximately 100
to 150 niotes (equivalent of 50 to 75 kg) of ATP arc synthesized daily. ATP is synthesized
mainly in oxidative metabolism and requires on average 27 moles of oxygen per day.
A human will typically use up his or her body weight of ATP over the course of the day.
In cellular energy metabolism, each ATP molecule is recycled (undergoes a process
of hydrolyzisand synthesis) 500-750 limes a day, which characterizes the intensity of
ATP metabolism in the body.
The ATP-ADP cycle as a process by which cells can store and release energy for
short-term use. The ATP-ADP cycle is the continuously ongoing energy recycling,
through oxidative phosphorylation of -flow energy> adenosine diphosphate (AD Pl to
*high energy* adenosine triphosphate (ATP), and the subsequent hydrolyzis of ATP
molecules back to A DP. in which energy stored in high-energy phosphate bond of
ATP is released and used in various cellular needs (Fig. 5.2).

Fig. 5JL ATP-ADP cycle

Thermodynamics of energy metabolism. From thermodynamics point of view living

organisms arc open systems. This means that there is an energy exchange between
system and environment. Every organic substance that enters into the organism is
characterized by internal energy. The portion of this internal energy that is available
for performing a thermodynamic work is called free energy, or Gibbs free energy (G).
When chemical reaction occurs al constant temperature, the change of free
energy (AG) and change of entropy (AS) is determined by the enthalpy change (AH),
reflecting the kinds and numbers of chemical bonds broken and formed, and the
entropy change, AS, describing the change in the system's randomness:
218 Chapter 5. Catabolism ana ceituiar bioenergetics

AG = AH—T x AS.
where T is the absolute temperature.
According to the second law of thermodynamics, for systems reacting at fixed
temperature and pressure, there is a tendency to achieve a minimum of the Gibbs
free energy A chemical reaction will proceed spontaneously if the AG is negative and
is accompanied by a reduction of free energy. These reactions arc exergonic and
thermodynamically favorable. If an absolute value of AG is high, the reaction goes
to the end and is considered as non-reversible. If AG is positive, the reaction is not
thermodynamically favored and will not proceed spontaneously {endergonic reaction).
In this case, reaction can only proceed with energy inputs from other energy sources,
such as the Sun in photosynthesis, or anol her exergonic reaction. I n biological systems,
thermodynamically unfavored reactions can only proceed if they arc «couplcd» with
exergonic reactions. Such reactions arc energetically coupled. The majority of such
reactions proceed with the participation of ATP. which acts as a coupling factor.
Metabolic pathways The Sun is the source of all the energy in the living cells.
The Sun’s energy reaches Earth and is used for photosynthesis by plants and algae to
produce glucose, which can be used to make ATP or be stored as starches. Plants then
arc consumed by herbivorous animals and insects, which can break down the starch
for energy or store it as glycogen, fats, or proteins. Carnivorous animals can then cat
these animals so that they can also produce fat, glycogen, or proteins in a chain of
biochemical reactions — metabolic pathways (Fig. 5.3).
Metabolic pathways can be categorized into two major categories. Anabolic
pathways build complex molecules from simpler ones and need an input of energy.
Photosynthesis in plants, fatty acid synthesis, or biosynthesis of proteins from amino
acids arc examples of anabolic reactions. Energy from the Sun is fixed and stored
in chemical bonds of energy-rich complex molecules, such as glucose, amino acids
and fatty acids (metabolic fuels). Therefore, anabolism is the biosynthesis phase of
metabolism in which smaller simple precursor is converted to the large and complex
molecules of the cell.
Catabolic pathways involve the breakdown of complex molecules into simpler ones
and typically release energy. Metabolic fuels arc metabolized in a series of oxidation
reactions in specific catabolic pathways and energy is released (Fig. 5.3, Table 5.1).
Some of this energy is dissipated as heat and used to maintain a constant temperature
of human body. The rest energy can be used to synthesize ATP. the ultimate energy
currency in the cell. Therefore, catabolism is a degradation phase of metabolism in
which large molecules are converted into smaller and simpler molecules. Catabolic
pathways typically consist, of two phases: first, hydrolyzis reactions, in which the large
molecules are broken down to the smaller molecules with the release of energy (the
examples arc breaking down of polymers into monomers during the food digestion:
starch into glucose, proteins into amino acids or triglycerides into fatly acids): and
second, oxidation reactions that involve the removal of hydrogen or electrons from
the product molecules of the first phase reactions. Anabolic and catabolic pathways
are precisely coordinated through multiple feedback and control mechanisms that
include transcriptional regulation, enzyme modifications, allosteric interactions,
com part men ration and metabolic specialization of organs.
5.1. Energy Producing and Energy Uhlizing Reactions. TH e ATP - ADP Cycle. Specific... 21 g

E
PROTEINS STARCH .2
I

Glycose Glycerol Fatty acids 5

Specific catabolic
pathways

Acetyl~CoA

Oxaloacetate s. Citrate
I
>1
<0
Malate Isocitrate B
TCA cycle
2h
2.
2H Fumarate (i-Ketoglutarate
ATP ADP CO2*t^------------- 2H
2
Succinate - - SuocinnyWCoA
s
NADH c
E
NADH dehydrogenase ADP + Pi 0
2H £
(Complex I) ATP E
0
o

Cytochrome bci ADP + Pi u

(Complex III) ATP L

Cytochrome c oxidase ADP + Pi

(Complex IV) ATP

2e- + 2H+ + 1TO2—H2O

Fig. 5.3. Conversion of energy stored in metabolic fuels into chemical energy of ATP in a chain ol
biochemical reactions of food digestion, specific metabolic pathways, tricarboxylic acid cycle (TCA
cycle) and electron transport chain (ETC)

Specific catabolic pathways

Glycolysis is a sequence of reactions in the cytosol that converts one molecule
of glucose into two molecules of pyruvate with the concomitant generation of twro
molecules of ATP in the substrate-level phosphorylation reactions, and two molecules
of NADH. This process is discussed in detail in section 6.2.
220 Chapter 5. Catabolism ana cellular bioenergetics

TaUle 5.1. ATP formation in me aerobic oxidation of glucose via glycolysis. me pyruvate dehydrogenase
complex reaction, me TCA cycle and oxidative phosphorylation (Lehniisger, Principles of Biochemistry)

Glucose ® glucose-6-pliosphate -1 ATP -1 Glycolysis

Fructose-6-phosphate ® fructose 1,6-phosphate -1 ATP -1

2 Glyceraldehyde 3-phosphate ® 2 2 NADH 3 or 5*

1,3-bia hasp hog lycerate

2 1,3-biphasphag lycerate ® 2 3-phosphogfycerate 2 ATP 2

2 Phasphoenolpyruvate ® 2 pyruvate 2 ATP 2

2 Pyruvate ® 2 acetyl-CaA 2 NADH 5 Pyruvate

decarboxy­
lation

2 Isocitrate ® 2 a-ketaglutarate 2 NADH 5 TCA cycle

2 a-Ketoglutaraie ® 2 succinyt-CoA 2 NADH 5

2 Succinyl-CaA ® 2 succinate 2 ATP or2GTP 2

2 succinate ® 2 fumarate 2 FADH? 3

2 Malate ® 2 oxaloacetate 2 NADH 5

Total 30-32

■ Calculated as 2.5 ATP per NADH and 1.5 ATP per FADH2. A iwgative value indicates consumption.
1 This number is eilher 3w5, depending on die mechanism used to shuttle NADH equivalents From the cytosol to the
rndochondriil matrix; tec me malate-aspartate shuttle antf me glycerol 3-ptiosphate shuttle.

Oxidation of fatty acids — ^-oxidation pathway. A different pathway exists

in mitochondria that degrades fatty acids. Carnitine transports fatty acids into
mitochondrial matrix through specific transporters, where they arc degraded toacctyi-
CoA in the reactions of p-oxidation of fatty acids. The acetyl-CoA then enters the
citric acid cycle. Alternatively, acetyl-CoA can give rise to ketone bodies. The FADH3
and NADH formed in the p-oxidation pathway transfer their electrons to O. through
the electron-transport chain. Oxidation of fatly acids is described in section 7.6.
.Amino acid catabolism. I n the cell, amino acids can be used for protein biosynthesis
or excess is converted to glucose or fatty acids. First, the amino groups from amino
acids are removed in the transamination reaction. In this reaction, NH3' group
is transferred to a common acceptor for amino groups — a-kcioglutaratc to form
glutamate and then ultimately is removed from the body. Then carbon backbones of
amino acids (a-ketoacids) arc convened to acctyl-CoA or the TCA cycle metabolites
that eventually arc oxidized to CO. and water. Amino acid catabolism can. potentially
contribute to some ATP synthesis through, the TCA cycle. However, only 10-15% of
bulk energy production in the cell can be attributed to amino acid catabolism. Sec
sections 8.6.8.7 and 8.8 for more details.
5.2. a Common Pathway for the Final Oxidation of Metabolic Fuels 221

52. A COMMON PATHWAY FOR THE FINAL OXIDATION

OF METABOLIC FUELS
The common catabolic pathway starts from the stage of pyruvate formation.
Of the large number of starting compounds, only two arc formed — pyruvate and
acetyl-Co A. The process that begins with the oxidation of pyruvate is called the
common catabolic pathway and includes oxidative decarboxylation of pyruvate and
TCA cycle, which is connected with ETC. Il is in the general path of catabolism,
where the main substrates for dehydrogenation reactions arc formed and finally
are oxidized to CO, and water. Together with the electron transport chain and
oxidative phosphorylation, the common, catabolic pathway is the main source of
energy in the form of ATP (Fig. 5.3, Table 5.1).
Oxidative decarboxylation of pyruvate is catalyzed by pyruvate dehydrogenase
complex (PDH), a huge multienzyme complex (Fig. 5.4) that contains three types
of enzymes: Pyruvate decarboxylase (E(X Dihydiolipoyl transacetylase (E3) and
Dihydrotipoamid dehydrogenase (Ep (Fig. 5.5 A). Additionally, PDH contains five
coenzymes: thiamine pyrophosphate (TPP), lipoic acid, FAD, NAD\ and CoASH
(Fig. 5.5 B).

Fig. 5.4. Structure of pyruvate dehydrogenase complex (PDH). licensed under the Creative Commons
222 Chapter 5. Catabolism ana ceituiar bioenergetics

A
Pyruvale
TPP, dehyckogenasa
lipoate, complex
S-CoA
. fad irreversibly
Pyruvate dehydrogenase converts
pyruvate to
complex (Ei + E2 + Eg}
CH3 ACfltyl-CoA,
a main fuel for
Pyruvate (Acetyl-CoAj the TCA cycle

A G n = -33.4 kJ/mol

Lip = Lipoamide
dehydrogenase complex

Fig. 5.5. Pyruvate cfecarboxylalio 11 reactions

► A. Pyruvate decarboxylase (Ej) is the first enzyme and uses thiamine

pyrophosphate (TPP) as a coenzyme, a derivative of vitamin Br The enzyme
catalyzes the cleavage of the carboxyl group as CO3 and the addition, of an acetyl
residue to the TPP. E, is composed of 120 polypeptide chains (30 tetramers)
(Fig. 5.4).
► B. Dihydrolipoyl transacetylase (E7) is the second enzyme of the complex. It
catalyzes the oxidation of the hydroxycthyl group and the transfer of the acetyl
group to lipoic acid, and then to CoASH Lo form acetyl-CoA. E2 is composed of
180 polypeptide chains (60 tri mens) (Fig. 5.4).
Thus, two coenzymes arc involved in this reaction: lipoic acid, which is firmly
linked to the enzyme, and coenzyme A. which is combined with the enzyme
at the time of the reaction. Hydrogen remains bound to lipoic acid, which is
converted to dihvdrolipoatc.
5.3. Tricarboxylic Acid Cycle (TCA cycle) 223

t C. Dihydrolipoamide dehydrogenase (E,) catalyzes die dehydrogenation of the

reduced lipoic acid and the transfer of hydrogen to the flavin adenine di nucleotide
(FAD)* which is tightly bound to the enzyme, and then to its second coenzyme
NAD', which is included in the complex only during the reaction. is composed
of 12 polypeptide chains (6 dimers).
The main products of PDH reactions arc CO2 NADH + H and acetyl-CoA.
NADH + H is further oxidized in the respiratory chain, where the energy is used to
synthesize 2.5 moles of ATP. and acetyl-CoA is oxidized in the TCA cycle. The PDH
complex is located on the inner mitochondrial membrane and is associated with it
from the matrix side.
Regulation of PDH. The PDH step is irreversible; as a result, animals arc not able
to synthesize glucose from acetyl-CoA. and therefore from fat. PDH is regulated
allostcrically and by protein phosphorylation and dephosphorylation by PDH
kinase and phosphatase, respectively. Phosphorylation of PDH turns olf its activity,
dcphosphorylation results in activation. High concentrations of ATP. NADH and
acetyl-CoA stimulate PDH kinase, while insulin, NAD , and ADP stimulate PDH
phosphatase. PDH is inhibited by acetyl-CoA which acts on E. (dihydrolipoyl
transacetylase), and NADH. which acts on E^ (dihydrolipoyl dehydrogenase).

5.3. TRICARBOXYLIC ACID CYCLE (TCA CYCLE)

The tricarboxylic acid cycle (TCA cycle), also known as citric acid cycle or the
Krebs cycle — is a series of biochemical reactions used by all aerobic organisms to
release stored energy through the oxidation ofacetyl-CoA derived from carbohydrates,
fats, and proteins, into ATP (Fig. 5.6). In addition, the cycle provides precursors of
some amino acids, as well as the reducing agent NADH. In eukaryotic cells, the citric
acid cycle occurs in the matrix of the mitochondria, while in bacteria it occurs in the
cytoplasm.
The TCA cycle is a central metabolic pathway that connects carbohydrate, fat, and
protein metabolism. The reactions of the cycle are carried out by eight enzymes that
completely oxidize acetate (a two-carbon molecule), in the form of acetyl-CoA, into
two molecules each of carbon dioxide and water.
The citric acid cycle begins with the transfer of two-carbon acetyl group from
acetyl-CoA to the four-carbon oxaloaceLatc to form six-carbon citrate (Fig. 5.7). The
reaction is catalyzed by citrate synthase. Citrate synthase is the firstand rate-limiting
enzyme of the TCA cycle and plays a key role in regulating energy generation.
Next, in the second reaction olT he TCA cycle the citrate is transformed to isoci irate
in the two-step reaction, which is catalyzed by aconitase (Fig. 5.8).
Then, in the third reaction of the TCA cycle, the isocitratc undergoes of
oxidative decarboxylation, producing a-kctoglutaratc and CO.,. This is also a two-
step process, which involves oxidation of isocitratc to oxalosuccinatc, followed by
the decarboxylation of the carboxyl group, forming a-kclogintaratc and NADH
(Fig. 5.9). The reaction is catalyzed by isocitratc dehydrogenase isoform 3 (IDH3).
Other iso forms of dehydrogenase ID Hl and IDH2 arc localized in. the cytosol and
peroxisomesand use NA DP" as coenzyme.
224 Chapter 5. Catabolism ana cellular bioenergetics

COO- COO“

Fig. 5.6. Tricarboxylic Acid cycle

H2O CoA-SH
CHg—C ■+■ O=C
. _ — COO" < J.
Citrate synthase
S-COA
ch2— coo- CH2~ COO"

[Oxaioacetate] C trate |
Acetyl-Co A
G’ = -32.2kJ/moi

Fig. 5.7. Citrate synthesis is a first reaction in the TCA cycle

5.3. Tricarboxylic Acid Cycle (TCA cycle) 225

CH-r= COO CHt—COO

H3U I
C—COO H—C—COO
I I
c— coo AcurnLisci HO—C—H

H COO

[as-Aconitatel [ Isocrtfate-I

£ G“= T3.3k*Mnwi

Fig. 5.8. Formation of isocilrate in the second reaction of the TCA cycle

-
COO" COO- coo- COO"
1 1 1
. 2 fl COa GHj. CHn
r z
1
H— C— C. z V }
—N, ,Z „
I
H—C
fsotitraie Ub. °-
HQ—C—n defrydrogenase c=o
< 1 1

✓V ® Zvz © /v

[i&odtraie Oxatosuccinate | [a-KetoglU!arale!

Fig. 5 J. Oxidative decarboxylation of isocitrale and formation ot ti-keto glularate in the third reaction
of the TCA cycle

The fourth reaction of the TCA cycle is a conversion of a-kctoglutaratc to

succinyl-CoA and formation of NADH. The reaction is catalyzed by a-kctoglutanitc
dehydrogenase complex (Tig. 5.1.0). Much like pyruvate dehydrogenase complex
(PDH), th is enzyme forms a complex composed of three components: a-kclogiularate
decarboxylase (Ej), dihydrolipoyl succinykransfcrase (E2) and dihydrolipoyl
dehydrogenase (E1) and contains coenzymes thiamine pyrophosphate (TPP), lipoic
acid. CoA. FAD and NAD. This reaction proceeds in three steps: (a) decarboxylation
ofa-ketoglutarate, (b) reduction of NAD+ to NADH, and (c) subsequent transfer to
CoA, which forms the end product, succinyl-CoA.
CH COO" ch2—cocr
I CoA-SH I
CH; '. NAD’ NADH CHj +CO;
\L j .
4

n-Ketoglutarale 1
-8

uehydfooenase C
complex
fa-Ketoglutarate) [ Succinyl-CoA )

A G’a = -33.5 kd/mot

Fig. 5.10. Conversion of a-ketogluiarate to succmyl-CoA and formation of NADH in me fourth reaction
of the TCA cycle
226 Chapter 5. Catano lism ana cellular bioenergetics

[n the fifth reaction of the TCA cycle, the succinyl-CoA is convened into
succinate (Fig. 5.11). The reaction is reversible and is catalyzed by succinyl-CoA
synthetase (SCS). SCS is the only enzyme- in the TCA cycle that catalyzes a reaction
in which a nucleoside triphosphate (GTP or ATP) is formed by substrate-level
phosphorylation. In the succinyl-CoA synthetase reaction, the high-energy bond
between HS-CoA and the succinyl group is hydrolyzed, and the energy released js
enough for synthesizing GTP from GDP + {Ph The GTP. from the energetic point
of view, is equivalent to ATP. In fact, GTP can transfer the (P) group to A DP to form
ATP. Since ATP can be produced from this reaction, without the participation of the
respiratory chain, this process is called Substrate Level Phosphorylation, in contrast
to the Oxidative Phosphorylation, where ATP synthesis uses energy generated in the
Electron Transport Chain.

COO’

CH?

COO’

Substrate-/eve/
p/rasp/Nyy/abon

Fig. 5.11. Succiiiyl-CoA syiUftesis and substrate-level phosphorylation

Substrate-level phosphorylation reactions arc minor pathways of AT P synthesis.

Substrate-level phosphorylation reactions occur outside of oxi dative phosphorylation
which is described below. Oxidative phosphorylation is the main pathway of ATP
synthesis under aerobic conditions.
In the sixth reaction of the TCA cycle, succinate is oxidized to fumarate (Fig. 5.12).
The reaction is catalyzed by succinate dehydrogenase (SDH). This reaction occurs in
the inner mitochondrial membrane. It is the only enzyme that participates in both the
TCA cycle and the electron transport chain. SDH will be discussed in more detail in
section 5.4 — Oxidative Phosphorylation.
5.3. Tricarboxylic Acid Cycle (TCA cycle) 227

COO" COO"
I Succinate I
H-—C-—H dehydrogenase H— C
I
H—G—H < "A ’ C —H
I -Ad FADHjj I
COO" COO"

(Succinate) (Fumarate]

Fig. 5.12. Sixth reaction ol TCA cycle - convers ion of s uccinate to fumarate by succinate dehydrogenase

The seventh stop of the TCA cycle, the reversible hydration of fumarate to malate
is catalyzed by fumarase ( Fig. 5.13). There arc two forms of fumarase, mitochondrial
and cytosolic. The mitochondrial isoenzyme is involved in Lhe TCA cycle, and the
cytosolic isoenzyme is involved in Lhe metabolism of amino acids.
h cc

II Fumarase
C*- OH
-OOC ^ \
H

^urnarate Carbanion | M alate J

transition state

£ G>=-3.B kJ/mol

Fig. 5.11 Conversion ol fumarate to malate by fumarase through a carhanion intermediate

The final, eighth reaction of TCA cycle is the reversible conversion of malate to
oxaloacctale that is catalyzed by malate dehydrogenase (MDH ). This reaction occurs
through the oxidation of hydroxyl group on malate and reduction of NAD* with
formation of NADH (Fig. 5.14). There arc two major isoforms of MDH in eukaryotic
cells. M DH, is found in the mitochondrial matrix, participating as a key enzyme in the
citric acid cycle that catalyzes the oxidation of malate. The other, MDH), is found in
the cytoplasm and participates in the ma late-aspartate shuttle.
COO" COO'
I MAD* NADH - H* I
HO—C—H O—*C
I
H~ C—H H— G—H
I
Malate
I I
dehydrogenase
GOO" COO"

L-MaJate ] [ Osalciacetate)

£ G’*= 29.7 kJi’mol

Fig. 5.14. The oxidation of malate Id oxaloacetate is coupled with NADH production and is catalyzed
by malate dehydrogenase (MDH)
228 Chapter 5. Catabolism ana ceituiar bioenergetics

Regulation of TCA cycle

The rate of the TCA cycle is precisely tuned to meet the cell's needs for ATP. The
citric acid cycle is regulated primarily by the concentration of ATP and NADU. The
key control points are:
► pyruvate dehydrogenase:
► isocit rate dehydrogenase :
► a-Kctoglutarate dehydrogenase:
► citrate synthase.
NADH, a product of all dehydrogenases in the citric acid cycle, inhibits pyruvate
dehydrogenase, isocit rate dehydrogenase, a-ketoglutarate dehydrogenase, and also
citrate synthase. Acetyl-CoA inhibits pyruvate dehydrogenase, while succinyl-CoA
inhibits a-kctoglutaratc dehydrogenase and citrate synthase. ATP inhibits citrate
synthase and a-kctoglutaraic dehydrogenase: however, ATP levels do not change
more than 10% in vivo between rest and vigorous exercise.
Calcium is also serving as a regulator in the TCA cycle. Calcium levels in the
mitochondrial matrix can reach up to the lens of micromolar levels. It activates
pyruvate dehydrogenase phosphatase, which in turn activates the PDH complex.
Calcium also activates isocit rate dehydrogenase and a-ketoglutarate dehydrogenase.
This increases the reaction rates of many of the steps in the cycle, and therefore
increases flux throughout the pathway. Note that calcium is an important regulator in
muscle, cells: the muscle contraction cycle is triggered by calcium ions.

Amphibolic functions of the TCA cycle

The term amphibolic is used to describe a biochemical pathway that involves
both catabolism and anabolism. The TCA cycle is a good example of an amphibolic
pathway because it consists of both the catabolic and anabolic reactions. The first
reaction of the cycle, in which oxaloacctatc (a four-carbon compound) condenses
with acetate (a two-carbon compound) to form citrate (a six-carbon compound) is
typical anabolic process (Fig. 5.7). Third and fourth reactions, conversion of isocit rate
to a-kciogluiaratc followed by its conversion to succinyl-CoA, arc catabolic (Fig. 5.9
and 5.10). A carboxyl group (-COO-) is lost in each step and eventually a four-carbon
compound succinate and 2 molecules of CO. arc produced. Intermediates of TCA
cycle can be used to synthesize other metabolites, such as amino acids, e.g., glutamate
and aspartate. For example, glutamate and glutamine can be synthesized from
a-kctoglutaratc: oxaloaccrate can be converted to aspartate and then to asparagine
by enzymes transaminase and asparagine synthase: pyruvate can be converted into
alanine by the enzyme alanine transaminase (Cahill cycle): pyruvate can be: converted
to oxaloacctatc by pyruvate carboxylase. The last reaction replenishes intermediates of
the TCA cycle and is referred asanaplerotic reaction.
There is a peculiar difference in the coenzymes used in catabolic and anabolic
pathways; in catabolic processes, NA D' serves as an oxidizing agent when it is reduced
to NADH. Whereas in anabolic reactions, the coenzyme NAD PH serves as the
reducing agent and is converted to its oxidized form NADP . NADP' (nicotinamide
adenine dinuclcotide phosphate) is a cofactor that is used in anabolic reactions, such
as lipid and nucleic acid syntheses, which require NA I) PH as a reducing agent.
5.4. Mitoctiondfial Electron Transport Chain and Oxidative PnospriDelation. Tnermogenesis 229

The cellular signaling system determines whether the amphibolic pathway will
function as an anabolic or catabolic mode by enzyme-me dialed regulation at a
transcriptional and post-transcriptional levels. As many reactions in amphibolic
pathways are freely reversible or can be bypassed, irreversible steps that facilitate
their dual function, arc necessary. The pathway will use a different enzyme for each
direction for the irreversible steps, allowing independent regulation of catabolism and
anabolism.
Overall, at the end of glycolysis, we have two pyruvate molecules that have plenty
of extractable energy. From a single molecule of glucose two molecules of pyruvate
arc generated and then two molecules of acetyl-CoA are produced along with two
molecules of NA DEI and two molecules of CO,. Two molecules of acetyl-CoA enter
the TCA cycle. In a single turn of the TCA cycle, three molecules of NADH and one
molecule of FAD H, a re generated from a single molecule of acetyl-CoA. Additionally,
one molecule of ATP or GTP is generated in the substrate-level phosphorylation
reaction (fifth reaction of the TCA cycle). Electrons of reducing equivalents NADH
and FADH,arc then transferred to the electron transport chain, where more ATP
molecules arc synthesized in the oxidative phosphorylation reactions.

5A MITOCHONDRIAL ELECTRON TRANSPORT CHAIN

AND OXIDATIVE PHOSPHORYLATION. THERMOGENESIS
Electron transport and oxidative phosphorylation in mitochondria arc final steps in
the conversion of energy of organic substrates (sugars, fat, or amino acids) into high-
energy phosphate bonds of ATP. the process that is called catabolism of metabolic
substrates.
The catabolism of organic compounds in the cells is accompanied by the
consumption ofoxygen and the release of CO... This process is call cd cellular respiration.
Oxygen in this process is used as a hydrogen acceptor from oxidizable (dehydrating)
substrates, as a result of which water is generated. The summary reaction of substrate
oxidation can be expressed as:

sh2+ko3*s+h.o,
where S is a metabolic substrate.
The energy released during the oxidation, reactions is partially spent on the
phosphorylation of ADP to form ATP. Overall, the body converts about 40% of
the energy released during oxidation into the energy of high-energy ATP bonds. A.
significant part of energy is dissipated in the form of heat.
The dehydrogenation reaction and the way by which the released energy is convened
to chemical bonds of ATP arc energetically coupled reactions. A set of complexes that
transfer electrons from electron donors to electron acceptors via consecutive redox
reactions is called an electron transport chain (ETC), or a respiratory chain (RC)
(Fig. 5J5 A and B). A process of synthesis of ATP from ADP in the ETC is called
oxidative phosphorylation
230 Chapter 5. Catabolism ana cellular bioenergetics

ATP

Matrix ADP+P,

Malaw Mjio^wndfial marrii

itYier
mwctKh-Kliial
menbcjie

jniermerntrane fll-T
Sfiace ------------- 1--------
FtaUEtione Aniirr.yCtn A
AmyQI

Inteffnentwarte space

Fig. 5_15. A Electron transport chain (ETC) and oxidative phosphorylation - simplified diagram.
Fe-S — iron sulfur centers of proteins. nH+ indicates that number of protons are pumped from the
matrix to the cytosol side. The return of protons through me ATP syntnase pore drives ATP synthesis,
mnibitors of ETC complexes are framed and sites of their action are shown (T). B. More detailed
diagram of milocrmdriai electron transport chain: Com ptexes I, IIJII an d IV are components of electron
transport chain located in the inner membrane of mitochondria. Complexes I, ill and IV transport
protons (H'J across inner mitochondrial membrane and generate a transmembrane electrochemical
gradient lAjdTt a motive force for ATP synthase. Complex ll — succinate dehydrogenase does not
participate in generation of electrochemical gradient Cytochrome c(Cyt c) is a low molecular weight
heme-containing protein, possessing nigh mobility in the lipid layer of the mitochondrial membrane.
Coenzyme Q (Q) is a non-protein electron carrier. FeS proteins contain n on-heme iron and are part
of comptexes I, II and ill. ATP synthase sometimes is called complex V and catalyzes ATP synthase
5.4. Mitoctiondrial Electron Transport Chain and Oxidative PnospriDelation. Tnermogenesis 231

Intermediate carriers in the ETC in higher organisms are coenzymes: NAD'

(nicotinamidc-adcninc di nucleotide), PAD and FMN (flavin adenine di nucleotide
and flavin mononucleotide), coenzyme Q (CoQ), the family of heme-containing
proteins — cytochromes (referred to as cytochromes, c;i c, u. t/T) and proteins
containing non-he me iron. All components in. this chain, arc organized into four redox
complexes connected by ubiquinone (CoQ) and cytochrome e (Fig. 5J5). Electrons
from the donors, such as NADH and FADH2, pass through the ETC to oxygen, which
finally is reduced to water. The ETC is comprised of series of electron donors and
acceptors. Each electron donor will pass electrons to a more electronegative acceptor,
which in turn donates these electrons to another acceptor, a process that continues down
the series until electrons are passed to oxygen, the most electronegative and terminal
electron acceptor in the chain. Each intermediate carrier initially acts as an electron
and proton acceptor and passes from the oxidized stale to the reduced form. Then it
transfers the electron to the next carrier and returns back to the oxidized state.
Four membra no-bou nd complexes have been identified in mitochondria. Three of
them arc proton pumps. The overall process of electron transport in ETC is depicted
below:

NADH+H* * Cb/npfex / - CoQ - Complex III - cytochrome c Comp/ex /(■ - H.O

t
Cbmp/e.x II
t
■Stfccfflorc

in I96I Peter Mitchell proposed the chcmiosmotic hypothesis, wrh.ich suggests

that most ATP synthesis in mitochondria comes from the electrochemical gradient
across the inner membranes of mitochondria by using the energy of NADH and
FADH, formed from the breaking down of energy-rich molecules such, as glucose
and fatty acids. The energy available in the electrons is used to pump protons from
the matrix across the inner mitochondrial membrane, storing energy in the form of a
transmembrane electrochemical proton gradient (ApH ). The protons move back across
the inner membrane through the channel in the enzyme ATP synthase. The flow of
protons back into the matrix of the mitochondrion via ATP synthase provides enough
energy for ATP synthesis. This radical hypothesis was initially received with the great
deal of skepticism in the scientific community. However, in 1978 Peter Mitchell was
awarded the Nobel Prize in. Chemistry.

Elements of electron transport chain

Proleins containing non-heme iron. A certain number of iron atoms in mitochondria
is not connected in the cytochrome heme, but form iron-sulfur clusters (Fc-S clusters).
These proteins arc also called iron-sulfur cluster proteins, or Fc-S cluster proteins,
because iron atoms arc bound to sulfur atoms of cysteine residues (Fig. 5.I6). Proteins
containing non-heme iron are involved in electron transfer in complexes I and 111.
232 Chapter 5. Catabolism ana cellular bioenergetics

A B

CyS — S S - Cys
<*-(§}„ ..Xs)-Cys

CyS-(s)'' ’’(s)-GyS
CyS - S rS — CyS

+ e-
Fe3+ " Fe2+
- e-

fig. 5.16. Structures of iron-suilur center A — Fe-S center; B — Fe2-S2 center; C — Fe4-S4
center. Cysteine residues (Cys) are involved in binding of Fe-S clusters in the protein. D —
oxidation and reduction Fe. Iron atoms can be in oxidized (Fe3*) or reduced (Fe?+) states

Coenzyme Q. Coenzyme Q|i( or CoQ]QS also known as ubiquinone, is a lipid-

soluble and contains quinone head and 6— IO isoprene repeats in the side chain
(number 10 in CoQ ll( refers to ten isoprene repeats in the side chain) (Fig. 5.17). The
CoQ is present in mitochondria of all respiring eukaryotic cells. CoQ serves as an
electron carrier in ETC between complexes l-III and ll-III. There arc three redox
states of CoQ: fully oxid ized (ubiquinone), partially reduced (semiquinone), and
fully reduced (u bi quinol). The capacity of this molecule to act as a two-electron
carrier (moving between the quinone and quinol form) and a one-electron carrier
(moving between the semiquinonc and one of these other forms) is a crucial to its
role in the electron transport chain. Biosynthesis is a major source of CoQ in the
cell. Some chronic diseases (cancer, cardiac diseases) or use of statins may decrease
CoQ biosynthesis and cause CoQ deficiency. In clinical practice, CoQ content is
measured in the plasma during a routine blood test.
5.4. Mitocnortarial Electron Transport Cnain and Oxidative Pnospnoryiation. Thermogenesis 233

Fig. 5.17. Chemical structure of coenzyme Q wim 5-10 isoprene repeats in the side cnain

Oh oh

NAD4-

FAD

Fig. 5.18. Coenzymes o! dehydrogenases. Structural formulas ol coenzymes NAD FAD and FMN
234 Chapter 5. Catabolism ana ceituiar bioenergetics

Flavin groups in FAD and FMN arc derived from riboflavin (vitamin U3)
(Fig- 5.18). The flavin group is capable of undergoing oxidation-reduction reactions
and can. accept electrons. Reduction is made with the addition of hydrogen atoms io
specific nitrogen atoms (Fig. 5.20).

Fig. 5.19. Chemical structure of heme cun cytochrome c)

Cytochrome c. Cytochrome c is a small heme-containing peripheral protein

that is loosely associated with the inner mitochondrial membrane from the
intcrmcmbranc space. Cytochrome c is a component of the electron transport chain
in mitochondria. The heme group of cytochrome c (Fig. 5.I.9) accepts electrons
from the complex 111 (the bCjComplex) and transfers electrons to the complex IV
(cy toe h rom c c ox id asc).
5.4. Mitochondnal Electron Transport Chain and Oxidative PnospriDelation. Tnermogenesis 235

Reduction
Oxidation

Reduction
Oxidation

f FAD or FMN]

Fig. 520. The redox reachoils of nicotinamide adenine dinucleotide (A) and flavin moieties of FAD
and FMN (B)

Complexes of mitochondrial electron transport chain (ETC)

► Complex I — NADH dehydrogenase;
► Complex II — succinate dehydrogenase;
► Complex III — the cytochrome b-c,complex;
► Complex IV — cytochrome c oxidase;
► Complex V — ATP synthase (F^FjATPasc).
Complex 1 (NADH dehydrogenase). Complex 1 is a very large enzymatic complex
catalyzing the first step of electron transport chain. The total number of sub units in the
human complex 1 enzyme is 45 (Fig. 5.21). Complex I contains 30 prosthetic groups.
Seven subunits arc encoded by mitochondrial genome. Total molecular mass of
complex I is over I million Dalton. The enzyme oxidizes NADH transferring electrons
to CoQ (ubiquinone). Of particular functional importance are the flavin prosthetic
group (FM N) and eight iron-sulfur clusters (Fe-S).
Complex I is involved in the formation of the transmembrane proton gradient with
a stoichiometry 4 H '/2c. The process begins with the transfer of protons and electrons
from NADH to ubiquinone (CoQ) and involves FM N and Fc-S clusters. The proposed
pathway for electron transport prior to ubiquinone reduction is as follows:

NADH-FMN-N.-N^-N.-N.-N^-N^-N.-CoQ,
where Nx is a label for iron sulfur clusters.
236 Chapter 5. Catabolism ana cellular bioenergetics

Ubiquinol (CoQH.,) diffuses in the membrane from complex I to complex III.

where it is oxidized to ubiquinone (CoQ). The How of electrons through Complex I
to CoQ is accompanied by the movemem of protons from the mitochondrial matrix
to the interment brane space, contributing to the generation of the transmembrane
electrochemical gradient.

Fg. 6.21. Mitochondrial Complex 1 Flow of electrons and protons in Complex I

Complex II (succinate dehydrogenase). Succinate dehydrogenase (SDH) is

composed of four subunits. Subunits SDH A and SDH B arc hydrophilic and are
facing mitochondrial matrix. SDH A contains a covalently attached flavin adenine
di nucleotide (FAD) cofactor and the succinate binding. SDH 13 contains three iron­
sulfur clusters. Subunits SDH C and SDH D are hydrophobic and arc anchored
to the inner mitochondrial membrane (Fig. 5.22). All subunits arc encoded by the
nuclear genome. Succinate dehydrogenase is the only enzyme that participates in
both, the TCA cycle and the electron transport chain. In step 6 of the citric acid
cycle, succinate dehydrogenase catalyzes the oxidation of succinate to fumarate with
the reduction of ubiquinone (CoQ) to ubiquinol (CoQHj. This occurs in the inner
mitochondrial membrane by coupling the two reactions together. Complex II is a
parallel electron transport pathway to complex I, but unlike complex I. no protons
arc transported to the intermem brane space in this pathway. Therefore, the pathway
through complex H contributes less energy to the overall electron transport chain
process.
5.4. Mitocnoodfial Electron Transport Chain and Oxidative PnospriDelation. Tnermogenesis 237

Succinate Fumarate

fad fadho
SDHA

3x[F&-SJ
Matrix
SDHB

SDHC SDHD

Inner mitochondrial
membrane
rn ' r. V'V'T1 IT Heme

inrermembrane space

Fig. 522. Mitochondrial Complex EL Schematic diagram of complex II m inner mitochondrial membrane.
SDH has two trans membrane subunits C. D and two units A, B, which are facing mitochondrial matrix

Complex Hl (cytochrome b — cJcomplex}. Complex III plays a critical role in

electron transport process. Complex III is composed of 11 subunits. These arc
respirator)' subunits: Cytochrome 6, cytochrome cr and Rieskc subunit, 2 core
subunits and 6 low molecular weight polypeptides. It has four cofactors: cytochrome
cytochrome f>, and a 2-Iron ferredoxin. Cytochrome & is encoded by mitochondrial
I) NA.
Complex III catalyzes the reduction of cytochrome c by oxidation of CoQ and
the concomitant pumping of 4 protons from the mitochondria! matrix to the
interment brane space. In the overall reaction 2 x QH, oxidized to Q; I x Q reduced
to QH-: 2 x Cyl c reduced: 2 x H' picked up from matrix and 4 x IT released into
intcrnicm brane space (Tig. 5.23). The summary reaction of complex III is shown
below:

QHn + 2 cytochromec(Fc3e) + 2H\j-*Q + 2cytochromec(Fe2') 4-4 H

The process by which the electrons arc transferred from the ubiquinol to
cytochrome c is known as the Q cycle. This cycle actually consists of two half-cycles.
In the first half-cycle. a ubiquinol molecule (CoQHJ attaches onto complex III and
transfers the two electrons to the complex. One of these electrons moves onto the
Rieskc subunit, then onto cytochrome c^and finally onto cytochrome c. Cytochrome
c, unlike ubiquinone, can only carry a single electron at any given time. The other
electron that comes from CoQH, follows a different pathway and moves through the
heme groups of cytochrome b and onto ubiquinone (CoQ) to form a partially reduced
unstable species called a semiquinone (CoQH-).
238 Chapter 5. Catabolism ana cellular bioenergetics

2H+

Fig. 523. Mitochondrial Complex III Sen emetic diagram of complex in in inner mitochonariai membrane
and electron flows in me Q cycle. As a result of Q cycle. 2 protons are taken from the matrix, 4 protons
are released into me imermemdrane space and two electrons are passed to cytoenrome c. Qo ana
Qi — Ubiquinol (Qty and ubiquinone (Q) Dinging sites of complex III, respectively

The two protons that were originally attached to ubiquinol arc transferred into the
intermembrane space. In the second half-cycle of the Q cycle, another ubiquinol
attaches onto complex 111. Upon binding, the two protons arc moved into the
intcrmcmbranc space and the two electrons follow the same pathways as before. The
electron that travels through the Ricskc subunit eventually ends up reducing a second
cytochrome c while the other electron travels onio the semiquinonc to form a fully
reduced quinone — ubiquinol (the ubiquinone must take up two protons from the
matrix to form the ubiquinol). Therefore, a single Q cycle reduces two cytochrome
c molecules, forms a single ubiquinol molecule, pumps four protons into the
intcrmcmbranc space and takes up two protons from the matrix.
Complex (V (cytochrome c oxidase). Complex IV is the last electron acceptor in
the ETC and is involved in the reduction of O-, to H20. Complex [V is a mu hi meric
complex (Fig. 5.24.), subunits of which arc encoded both nuclear and mitochondrial
genome. Al least 20 subunits arc present in human complex IV. Three subunits of
Complex IV arc encoded by mitochondrial genome. Composition and stoichiometry
of subunits and prosthetic groups depend on tissues and cell types. Additionally, over
30 of accessory chaperone proteins arc required for the assembly of functional complex
IV. Complex IV catalyzes the transfer of electrons from reduced cytochrome c to the
final acceptor of electrons, On, in a process that is coupled to proton translocation
and formation of a proton gradient across the inner mitochondrial membrane. The
summary reaction can be expressed as:
5.4. Mitoctiondnal Electron Transport Chain and Oxidative PnospriDelation. Tnermogenesis 239

4CytCM
intermemtsrane space
4H*

Fig. 5.24. Mitochondrial Complex IV. Schematic diagram of complex IV in inner mitochondrial
membrane and electron flows

Reductive potential or redox potential is a measure of the tendency of a chemical

species to acquire or lose electrons and thereby be reduced or oxidized, respectively.
Redox potential is measured in millivolts (mV). Each species has its own intrinsic
redox potential. The more positive the redox potential the greater the species1 affinity
for electrons and the tendency to be reduced.
hi the electron transport chain, the lowest potential is found in. NADH in complex
I (—320 mV)r keeping its position at the start of the transport chain. The redox
potential increases continuously along the respiratory chain to reach its highest value
at oxygen (4- 0.817 mV), which, therefore, has the highest affinity for the electrons
(Fig. 5.25).
ATP synthase. Human mitochondrial ATP synthase, sometimes also called the
complex V, consists of two functional domains: Fr situated in the mitochondrial
matrix, and F, located in the inner mitochondrial membrane (Fig. 5.26). The F
portion is a hydrophobic complex with a tetramer shape. The FDis a proton pore
that is embedded in the mitochondrial inner membrane. Human Foconsists of
three main subunits, a, b. and c, and six additional subunits, d. e. f. g. F6, and 8
(or A6L). Subunit a is encoded by mitochondrial DNA, while other subunits of
ATP synthase arc encoded by nuclear genes. Complex V uses the energy created by
the proton electrochemical gradient to phosphorylate ADP to ATP. Magnesium ion
(Mg34) is necessary for efficient ATP synthesis. The F, portion of ATP synthase is
hydrophilic and protrudes into the mitochondrial matrix space. Subunits ci and
p form a hexamer and three of them bind ADP. Other three subunits y, 6, and e
catalyze the ATP synthesis (Fig. 5.27).
240 Chapter 5. Catabolism ana ceituiar bioenergetics

60

Comptexes I II III IV

Fig. 5.25. Redox potentials, and The corresponding free energy levels, of electron carriers in me
respiratory chain (complex HV). Blue arrows show electron flow and red arrows snow proton
pumping. The TCA cycle provides NADH, wnicn is reduced to NAD + H* in complex I. The TCA cycle
also provides FADH?I and tn is is reduced to FAD + H* in complex n. The glycerol 3-phosphate snuttie
{G3P] shuttle provides FADH.. to coenzyme Q (CoQ). Reduced oxygen, which recombines wim protons
to yield water, is the end product of respiration

The energy generated by the flow of electrons through the respiratory’ chain,
is used for the coupling with phosphorylation of ADP. These two processes arc
interdependent: oxidation cannot proceed in the absence of ADP. The ratio of
oxidation and phosphorylation is determined by the P/O ratio, which shows how
much Pi is used for the formation of ATP during the conversion of one grant-atom
ofO, to H,0 (the number of moles of phosphorylated ADP per 1/2 mole of oxygen).
The P/O ratio is called the coefficient of oxidative phosphorylation and depends on
the entry point of reducing equivalents in the electron transport chain. For example,
for substrates oxidized by NAD-depcndent dehydrogenase, P/O =2.5, since there
arc three sites in the respiratory chain, where electron transfer is associated with
ATP synthesis. Not all substrates transfer electrons and protons to NAD; some arc
oxidized by FA D-de pendent dehydrogenases, which transfer protons and electrons
directly eo ubiquinone, bypassing complex I. In this case, P/O = 1.5. In reality, the
phosphorylation coefficient is always less than the theoretical value. because part of the
energy that is released during the transport of electrons is not spent on ATP synthesis,
bin to transfer substances through the mitochondrial membrane or disappeared as
heat.
5.4. Mitocnoodfial Electron Transport Chain and Oxidative PhospriDelation. Thermogenesis 241

Fig. 5 26 Mitochondrial ATP synthase, or complex V. Schematic drawing of ATP synthase. ATP synthase
consists of two functional domains, Fj and Fr F, comprises different subunits (three u, three p, and
one y, a and c) and is situated in the mitocnondriai matrix. Fd contains subunits c, a. d, a, F6, OSCP and
the accessary subunits e, f, g and A6L F, subunits y, 6 and c constitute me central stalk of complex
V. Subunits D. d, FE and OSCP form the peripheral stalk. Protons pass from die intermembrane space
to me matrix through Fa, which transfers the energy created by the proton electrochemical gradient
to F., where ADP is phosphorylated to ATP. One p subunit is taken out to visualize the central stalk

Supercomplexes. There is mounting evidence that, the enzymes of the

mitochondria! respiratory chain assemble into larger, supramol ocular structures
called respirasomes or supercomplexes. The most common supcrcomplcxcs
observed arc complexes I/l 11, complexes 1/II 1/IV, and complexes 111/IV. Most of
Complex IT is found in a free unbound form. Complex V' can be found as a part
of supcrcomplcxcs. I t has been hypothesized that the organization of respiratory
enzymes into supcrcomplcxcs reduces oxidative damage and increases the
efficiency of electron transport i n mitochondria.
Adenine nucleotide translocase. ADP/ATP translocase, or ADP/ATP carrier
transports .ATP synthesized from oxidative phosphorylation in the matrix into the
interment brane space and then to the cytoplasm, where it can be used as the principal
energy currency. ATP and ADP cannot cross the inner mitochondrial membrane due
to their high, negative charges, but ADP/ATP translocase, an ami porter, couples the
transport of these two molecules. Human cells express four different isoforms of ADP/
ATP translocases, and four distinct genes encode them.
242 Chapter 5. Catabolism ana cellular bioenergetics

2. Generation of ATP

Fig_ 527. Mechanism of ATP synthesis Uy ATP synthase (complex V}. Catalytic cycle of ATP synthase
involves 3 phases that occur in three different catalytic centers: 1 — binding of ADP and inorganic
phosphate (Pi); 2 — phosphorylation of ADP and generation of ATP; 3 — release of ATP

Inhibitors of ETC and un couplers

Complex I is inhibited by more than 60 di fie re nt families of compounds. Most
well-known and widely used is rotenone.
There are two types of inhibitors of complex II: those that bind in the succinate
binding site and those that bind in the ubiquinone binding site. Ubiquinone type
inhibitors include carboxin and ihenoyltrifluoroacelonc. Succinate-analog inhibitors
include the synthetic compound ma I onatc as well as the TCA cycle intermediates,
malate and oxaloacelatc.
Complex lit is specifically inhibited by antibiotic Antimycin A. Antimycin A
binds to complex III and inhibits the oxidation of ubiquinone, thereby disrupting the
Q-cyclo It also causes the disruption, of the entire electron transport chain.
Cytochrome c oxidase (complex IV) is reversibly inhibited by cyanide (CM ) and
carbon monoxide (CO). Cyanide and carbon monoxide bind to cytochrome c oxidase
heme a3-Cun binuclear center. Binding of cyanide or carbon monoxide to bi nuclear
5.4. Mitocnoodfial Electron Transport Chain and Oxidative PnospriDelation. Tnermogenesis 243

center prevents the transport of electrons from cytochrome c to oxygen, and as a result,
causes asphyxiation of cells, meaning that the cell can no' longer aerobically produce
ATP for energy and cell death, occurs rapidly. Cyanide is one of the most potent and
rapidly acting poisons known. The central nervous system is the primary target for
cyanide toxicity.
Nitric oxide (NO) also binds to the cytochrome c oxidase bi nuclear center, and
under physiological conditions, NO is thought to be an endogenous modulator of
oxidative phosphorylation.
II igher concentrations of molccular<JKygcn are needed to compensate for increasing
inhibitor concentrations. Oxygen therapy is often used in the emergency departments
as an. antidote for cyanide and carbon monoxide poisoning. Other ligands, such as
nitric oxide and hydrogen sulfide, can also inhibit cytochrome c oxidase by binding to
regulatory sites on the enzyme, reducing the rate of cellular respiration.
OLigomycin is an antibiotic that inhibits ATP synthase (complex V) by blocking its
proton channel ( F^ubunit), which is necessary for oxidative phosphorylation of ADP
to ATP. The inhibition of ATP synthesis would also slop electron transport chain.
Inhibitors of ETC complexes and the sites of their action arc depicted on the
Fig. 5.15, panel A.
ADP/ATP translocase is specifically inhibited by atraclylosi.de and bongkrckic
acid. The high affinity (Kd in the nano molar range) makes each inhibitor a deadly
poison by obstructing cellular respiration.
Hvpoenergetic state. Disruption of any stage of metabolism, leading to the
cessation of ATP synthesis, is fatal for the cell.
The state in which the synthesis of ATP is reduced is combined with the term
*hypoencrgctic*. The causes of hypocnergetic states can be fasting, deficiencies of
vitamins B,, PP+ Bp hypoxia. Hypoxia can occur with a lack of oxygen in the inhaled
air; in pulmonary diseases and impaired pulmonary ventilation; with circulatory
disorders caused by heart disease, spasm and vascular thrombosis, blood loss. The
causes of hypoxia can be hereditary or acquired defects of hemoglobin.
Genetic disorders caused by deficiencies of the TCA cycle and ETC. The TCA cycle is
crucial to the metabolism of living cells and any significant disruption of these reaction
systems causes an energy deficit in the cell. The TCA cycle enzymes arc all nuclcarly-
cncoded. Although very rare, inborn errors in the TCA cycle genes may be severe or
even be incompatible with life.
Conversion of pyruvate to Acetyl-CoA in PDH reaction and conversion of
a-keioglutaratc to succinyl-CoA in the fourth reaction of the TCA cycle requires
coenzyme thiamine (vitamin Bp. Thiamine is also important in the production of
NAD PH via the pentose phosphate pathway (section 6.4) and breakdown of branched
amino acids (section 8). Thiamine is a water-soluble vitamin that is not actively stored
in the body, and. therefore, must be obtained in the diet. Thiamine deficiency can
cause numerous disorders, such as Wernicke-KorsakofFs syndrome and beriberi
disease.
Aconitase deficiency is characterized by myopathy with severe exercise intolerance
in patients. Mutations in isocitratc dehydrogenase (1DH3) gene are associated with
various brain tumors and acute myeloid leukemia (AM L). Fumarate deficiency is
associated with severe neurological abnormalities, poor feeding, failure to thrive.
Recent studies suggest the importance of fumarase in DNA re pair path ways, suggesting
244 Chapter 5. Catabolism ana ceituiar bioenergetics

its tu mor-repress! ng. role. Citrate synthase deficiency may cause worsening of tumor
malignancy.
Genetic defects of ETC, Defects in ETC can be caused by mutations in. cither
mitochondrial or nuclear genes and cause mitochondrial diseases. Genetic diseases
affecting mitochondrial oxidative phosphorylation system are referred to as Oxphos
diseases.
Genetic defects affecting complex I include various neurodegenc native and
neuromuscular diseases, and Leigh syndrome.
Mutations in. genes that encode complex 11 subunits arc causative of various
disease, including Leigh syndrome, mitochondrial encephalopathy, optic atrophy,
numerous tumors (mostly benign), hereditary paraganglioma and hereditary
plicoc h romocy tom a.
Mutations in complex ill genes manifest as exercise intolerance, Ujomstad
syndrome and the GRACILE syndrome, which in neonates are lethal conditions.
Genetic defects in genes encoding cytochrome c oxidase subunits result in severe,
often fatal metabolic disorders. Cytochrome c oxidase deficiencies usually manifest in
early childhood and affect predominantly tissues with high-energy demands (brain,
heart, muscles). The vast majority of cytochrome c oxidase disorders arc linked to
mutations in nuclear-encodcd cytochrome c oxidase assembly proteins or chaperones.
Mitochondrial complex V deficiency can cause a wide variety of multi-organ
symptoms, particularly the nervous system and the heart. The disorder can be life-
ill rca ten ing in infancy or early childhood. Affected individuals may have feeding
problems, slow growth, low muscle tone (hypotonia), extreme fatigue (lethargy), and
developmental delay. They tend to develop elevated levels of lactic acid in the blood
(lactic acidosis), which can cause nausea, vomiting, weakness, and rapid breathing.
High levels of ammonia in the blood (hyperammonemia) can also occur in affected
individuals, and in some cases, result in abnormal brain function (encephalopathy)
and damage of oilier organs.

Ihermogenesis
The synthesis of ATP molecules consumes about 40-45% of the total energy of
the electrons transferred through the center of energy, about 25% is spent on work on
the transferor substances through the membrane. The rest of the energy is dissipated
in. the form of heat and is used by animals to maintain body temperature. Additional
heat generation can occur with the dissociation or uncoupling of respiration and
phosphorylation. Uncoupling of electron transport from oxidative phosphorylation
can generate additional heat, but will inhibit the ATP synthesis, without affecting ihc
respiratory chain.
The uncoupling of oxidative phosphorylation can be biologically beneficial, ft
allows to generate heal for maintaining body temperature in newborns, in winter
animals, and in all mammals in the process of adaptation to cold. The process is
called non-shivering ihcrmogencsis. In newborns, as well as winter-sleeping animals,
there is a special tissue that specializes in a heal production through the separation
of respiration and phosphorylalion — the brown faL, or brown adipose tissue (BAT).
The BAT cells contain lots of mitochondria. The mitochondria of BAT have a
5.5. Oxygen Toxicity 245

large excess of respiratory complexes compared to ATP synthase. Mitochondria in

the I? AT contain targe amount (about 10% of all proteins) of uncoupling protein-1
(UCP-I) — thermogen in. Thcrmogcnin acts as a biological uncouplcrand promotes a
heat generation al the expense of an ATP synthesis.
BAT is present in newborns, but its content is reduced in adults. Howe vol in recent
years, facts have appeared that small deposits of brown fat remain in various regions of
adult humans. Additionally, other uncoupling proteins have been discovered in various
tissues. Activation of thcrmogcnin promotes a weight loss and may be beneficial for
the organism. Non-shivering thermogenesis is regulated by the thyroid hormone and
the sympathetic nervous system. Some hormones, such as norepinephrine and leptin,
may stimulate thermogenesis by activating the sympathetic nervous system.
Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling
between the electron transport and phosphorylation reactions and thus inhibit
ATP synthesis without affecting the respiratory chain and ATP synthase (complex
V). Example of mitochondrial uncoupler is a proionophore (IT ionophore)
2.4-dinitrophcnol (DNP). Uncouplers arc weak hydrophobic acids which can
transfer protons from the intermembrane space though a hydrophobic layer of the
inner membrane back into the mitochondrial matrix, preventing the creation of
transmembrane proton gradient required for activity of ATP synthase. As a result of
uncoupling, the cellular concentration of ATP decreases and ADP concentration
increases. In this case, rates of oxidation of NADH and FADH, increase, as well as a
rate of oxygen consumption. However, energy is released in the form of a heal, instead
of ATP
Fatty acids also can act as u ncou plots. On the outer side of the inner mitochondrial
membrane, fatty acid anions bind protonsand transfer them to the inner mitochondrial
membrane through ADP/ATP translocase, reducing the transmembrane proton
gradient. In the matrix, protons arc dissociated from fatty acids and fatty acid anions
arc returned back to the outer side of the inner mitochondrial membrane.
Low temperature stimulates the rate of noradrenaline release from sympathetic
neurons. It loads to the activation of lipase in adipose tissue and the mobilization
of fatty acids. Free fatly acids serve not only as a metabolic fuels but are important
mediators for the uncoupling of respiration from oxidative phosphorylation in
mitochondria. There fore, fatty acids are important for the regulation of thermogenesis
and adaptation of the body Lo the cold environment.

5.5. OXYGEN TOXICITY

Reactive oxygen species (ROS) arc chemically reactive species containing oxygen.
Reactive oxygen species include superoxide anion <O£), hydrogen peroxide (H.Oj,
and hydroxyl radical (HO”). ROS are generated in the electron transport chain du­
ring mitochondrial oxidative metabolism as well as in cellular response toxenobiotics,
cytokines, and bacterial invasion.
The generation of ROS i n the cells occurs continuously. The structure of O, allows
its reduction by the addition of one electron at a time, leading to the generation of
oxygen radicals. A radical is a molecule with highly reactive unpaired electron in
an outer orbital, which can initiate chain reactions by removal of an electron from
246 Chapter 5. Catabolism ana cellular bioenergetics

another molecule to complete its own orbital. The stepwise transfer of elect tons to O3
results in the formation of superoxide anion (Op, then hydrogen peroxide (H3O3)T
and finally hydroxyl free radical (OH ’):
e _e.2H* e.rr e.H*
O2 H2O2^ H2O + 0H' 2H2O

The end products of this reaction sequence arc two molecules of water. The
hydroxyl radical is the most dangerous free radical because it is involved in reactions
such as lipid peroxidation and generation of other toxic radicals. Hydrogen peroxide
itself is not a free radical but is convened be the Fenton-, or Haber-Weiss reactions
io the hydroxyl radical in the presence of Fe3' or Cu . which are prevalent in cells.
In mitochondrial electron transport chain highest ROS producers arc Complex I
and Complex III. Leakage of electrons at complex I and complex 111 leads to partial
reduction of oxygen to form superoxide. Superoxide (O’) is the anionic form of O3
and is a free radical. Respiratory complex I leaks superoxide into the matrix and
respiratory complex III leaks superoxide into both the matrix and the intcrmembrane
space. Because of its negative charge, superoxide is unable to cross the mitochondrial
membrane. However, hydrogen peroxide (H3O2)t a product of SOD2 reaction (see
below), is released from mitochondria to the cytosol in proportion to the proton
potential and can be ultimately convened by enzyme catalase into H/) and ()..
Another free radical is nitric oxide (NO), which is both essential to life and toxic.
Al low concentrations, nitric oxide acts as neurotransmitter or as a hormone that
causes vasodilation. It is synthesized from arginine by nitric oxide synthases. At high,
concentrations nitric oxide binds to oxygen to form reactive toxic species that contain
both nitrogen and oxygen (RNOS). These free radicals arc also involved in damage of
the cells in chronic inflammatory diseases.
Cell damage caused by reactive oxygen species. Overproduction of ROS (oxidative
stress) causes damage to proteins, nucleic acids, lipids and is implicated in various
disease states such as atherosclerosis, diabetes, cancer, ncurodegc notation, and
aging. Oxygen radicals are also produced in certain cells during inflammation caused
by bacterial infection. To combat microbial infections, phagocytes produce toxic
oxygen radicals in a process known as the respiratory burst. The phagocytes then kill
the engulfed bacteria by the toxic radicals. Ingestion of drugs and chemicals, smog,
radiation can lead to the formation of reactive oxygen species. Damage from reactive
oxygen species often occurs during perfusion of the tissues with solutions containing
high concentration of O, as happens in patients who had an ischemic episode in which,
localized O. levels arc lowered due to blockage of an artery- and then have undergone
procedures to remove the blockage. Myocardial ischemia/reperfusion injury develops
as a result of the accumulation of reduced coenzymes NADH and FADI-iJn the
mitochondria at the absence of oxygen due to blockage of blood flow. After removing
the blockage oxygen supply to the tissues is restored as electron flow in FTC. Thus,
electrons leak easily from the ETC and more reactive oxygen species arc formed.
High levels of mitochondrial ROS activate apoptosis/autophagy pathways capable
of inducing cell death. On the other hand, small amounts of ROS arc necessary for the
cell and serve as critical signaling molecules in cell proliferation and survival.
5.5. Oxygen Toxicity 247

Reactive oxygen species and lipid peroxidation. Free radical chain reactions, which
form lipid free radicalsand lipid peroxides in membranes, make major contributions
to ROS-induced injury in the cells.
The radicals of polyunsaturated fatty acids in phospholipids of plasma and
organelle membranes are subjected to lipid peroxidation (Fig. 5.28). These free radical
chain reactions arc initialed by removal of hydrogen from polyunsaturated fatty acid
by hydroxyl radical COH'), thereby forming a lipid radical (L ). The free radical chain
reaction is propagated by reaction with O3 forming the lipid peroxy I radical (LOO ‘)
and lipid peroxide (LOO ‘). One of the main end products is malondialdehyde and
can be detected in the blood. Malondialdehyde is an active metabolite and forms the
cross-li nk between the amino groups of proteins and phospholipids thus causing the
disruption of membranes.

Fig. 528. Meefianrsm of lipid peroxidation (Young IS. McEneny J (2001). -lipoprotein oxidation and
atherosclerosis-. BiODh&m Soc Trans 29. DOE 10.1042/bst02 90358)

DNA and proteins arc also subjected to oxidative damage. In proteins, the amino
acids methionine, cysteine, arginine and proline arc the most susceptible to oxidative
damage. The damaged proteins appear in many diseases, particularly associated with,
aging. The pigment lipofuscin consists of a mixture of oxidized crosslinked lipids and
damaged proteins formed by reaction between malondialdehyde and amino acids
radicals. These pigment spots appear on the skin of the hands of elderly individuals
and arc considered as a hallmark of aging. In patients with Parkinsori s disease, the
lipofuscin granules appear in degenerating neurons as Lewy bodies.
Cellular defenses against oxygen toxicity. The cells arc protected from oxidative
damage by several mechanisms. Al first dietary antioxidants, such as vitamins E, Cd
248 Chapter 5. Catano lism ana cellular bioenergetics

flavonoids and endogenous antioxidant, such as urate, can terminate the free radical
chain reactions. The enzymatic defense against reactive oxygen species includes the
enzymes superoxide dismutase, catalase , and glutathione peroxidase.
Mammalian mitochondria arc well equipped to scavenge the superoxide generated
either internally or externally and defend cells from ROS-induced toxicity. The
manganese superoxide dismutase (SOD2) is located in the mitochondrial matrix,
where it protects mitochondrial targets against the internally generated superoxide.
SOD2 catalyzes the reaction that converts two molecules of superoxide (O1 ‘ ) to one
molecule of oxygen (O,) and one molecule of hydrogen peroxide (H^O):

2 HO3-O34H3O3,

where HO, is a protonated form of superoxide.

Two other mitochondria I enzymes arc also capable of efficient scavenging of
externally produced ROS: superoxide dismutase (SOD I) and cytochrome c plus
cytochrome c oxidase system.

Review test
I. Match the number on the figure with the letter.

A
ATP

Matrix ADP + P,

Maiale
T
Oligomycin

Cytosol T_ nH*

Rotenone
Amytai

Components of ETC:
A. Cytocromoxidase.
B. Cytochrome b-cl complex.
C. NADH dehydrogenase.
D. Succinate dehydrogenase.
E. Fumarase.
5.5. Oxygen Toxicity 249

2. Using the scheme of test L match she number of the enzyme on the figure of EEC with
the letter denoting the coenzyme of this enzyme.
Coenzymes:
A. Heme.
B. TMN
C. Heme, Cu 3'.
D. NAD .
E. FAD.
J. Match the figure and the letter.

Lip = Lipoamide
dehydrogenase complex

Enzymes:
A. Pyruvate carboxylase.
B. D i hydro! ipoa mid dehydrogenase.
C. Pyruvate dehydrogenase.
D. Dihydrolipoyl transacetylase.
E. Malate dehydrogenase.
4. The catabolic pathways. Match the figure and the letter
A. Common catabolic pathways.
B. Krebs cycle.
C. Oxidative phosphorylation.
D. Digestion.
E. Oxidative decarboxylation.
250 Chapter 5. Catabolism ana cellular bioenergetics

5. Match the number of the reactions wilb the letter denoting the enzyme.

SCc-A
5.5. Oxygen Toxicity 251

The reactions with, generation of ATP:

A. Succinyl-CoA synthetase.
B. a-kctoglularatc dehydrogenase.
C. Succinate dehydrogenase.
D. Citrate synthase.
E. Isocitrate dehydrogenase.

Situational Problems
1. Oxidative decarboxylation of pyruvate and the TCA cycle in muscles arc
stimulated by increased aerobic exercise. These processes operate only when
O2 is present, although oxygen docs not participate directly in these processes.
Explain why oxidative decarboxylation of pyruvate is activated under aerobic
conditions. For the answer:
a) describe the overall reaction catalyzed by the pyruvate dehydrogenase
complex (PDH) and its regulation:
b) outline the intermediates and enzymes of the TCA cycle;
c) explain the relationship between the reactions of PDH and the TCA cycle
and the respiratory chain.
2. A 4-year-old girl was diagnosed with thiamine deficiency and the symptoms
include tachycardia, vomiting, convulsions. Laboratory examinations reveal
high levels of pyruvate, lactate and (x-kcloglutaraic. Explain which coenzyme
is formed from vitamin B and its role in oxidative decarboxylation of pyruvate.
For that:
a) describe the structure of pyruvate dehydrogenase complex (PDH) and the
cofactors that it requires:
b) discuss the symptoms which are connected with the thiamine deficiency and
its effects on PDH and a-kc login taraie dehydrogenase complex:
c) explain the changes in the levels of mentioned metabolites in the blood:
d) name the described disease.
3. It is known that Leber hereditary optic neuropathy (LHON) is an inherited
form of blindness due to the missense mutations of mitochondrially encoded
proteins of complexes I or HI of the ETC. Explain why disorder in the structure
of these components of ETC can impair electron transport and oxidative
phosphorylation. For the answer:
a) draw the scheme of ETC and describe the structure of its components:
b) explain how the electrochemical gradient is gene rated during the transport of
the electrons;
c) cxplainc what the respiratory conLrol of the ETC and P/O ratio is.
4. Mitochondrial DNA (miDNA) encodes some proteins of the ETC. Point
mutations of mitochondrial genes can impair the electron transport chain and
oxidative phosphorylation. One of the syndrome resulting from disorders of
miDNA-cncoding protein is ME LAS syndrome (mitochondria! myopathy,
enccphalomyopathy, lactic acidosis and stroke). The earliest manifestations are
the neurological and muscular abnormalities due to the greater dependence of
brain, heart and skeletal muscle on mitochondrial ATP synthesis. Explain why
252 Chapter 5. Catabolism ana cellular bioenergetics

the levels of lactate and pyruvate arc increased in ME LAS syndrome. For the
answer:
a) discuss the respiratory control in ETC and significance of the ratio NADH/
NAD";
b) describe the reactions of oxidative decarboxylation of pyruvate;
c) explain why the disorders in the structures of components of ETC leads to the
activation of the reaction, which is catalyzed by lactate dehydrogenase.
5. To control the damage that rats may cause in the buildings, fiuoracetate (FA)
is used. After entering a cell FA is converted into very toxic fluoroacctyl-CoA.
The effect of FA was studied in the experiments on intact isolated rat hearts.
It was found after perfusion with FA the rat hearts the concentrations of the
TCA cycle metabolites were decreased. Only the level of citrate was significantly
higher than in controls.
a) describe the significance of the TCA cycle as the common catabolic
pathway and explain why blocking of this cycle leads to the lethal effect of FA:
b) indicate the reaction where the FA blocks the TCA cycle and Lhe enzyme
which is inhibited by FA:
c) explain why concentration of citrate is increased.
6. Red blood cells transport oxygen to the peripheral tissues, but they do not
produce ATP by oxidative phosphorylation.
a) indicate the final electron acceptor in the ETC;
b) indicate the location of ETC in the cells and draw the scheme of the ETC;
c) describe the mechanism of oxidative phosphorylation and explain why this
way of ATP synthesis is not possible in RBC.
7. In the brown adipose tissue (BAT) in the newborns, transmembrane proton
gradient is used not only to produce ATP, but to keep the newborns warm.
The mitochondria of BAT have a unique protein in their inner membrane —
thermogenin, also called the uncoupling protein I (UCPI). Explain the role of
UCPI in thermoregulation. For the answer:
a) draw the scheme of the ETC and explain how the electrochemicaJ gradient is
generated during the electron transport;
b) describe the mechanisms of uncoupling of oxidative phosphorylation by
UCPI;
c) compare the changes in the transmembrane proton gradient after the action of
inhibitors of lhe respiratory chain (including rotenone, antimycin A, cyanide,
carbon monoxide) and the uncoupling agents.
 Chapter 6
CARBOHYDRATE METABOLISM

6.1 Digestion, Absorption and Transport of Dietary Carbohydrates. Disorders of Digestion

and Absorption of Carbohydrates
6J. Glucose Catabolism. Aerobic Glycolysis,. Anaerobic Glycolysis and Complete Oxidation
of Glucose
6.1. Gluconeogenesis. Regulation of Glycolysis and Gluconeogenesis
6.4. Pentose Phosphate Pathway
6.5. Synthesis and Degradation of Glycogen
6.6. Regulation of Glycogen Metabolism
6.7. Glycogen Storage Diseases
6.8. The Metabolism of Ethanol in the Liver

6.1. DIGESTION, ABSORPTION AND TRANSPORT OF DIETARY

CARBOHYDRATES. DISORDERS OF DIGESTION AND ABSORPTION
OF CARBOHYDRATES
There are dozens of different monosaccharides and lots of di fie rent oligo- and
polysaccharides in a human body. Carbohydrates arc a part of live organisms and
together with proteins, lipids and nucleic acids determine the specificity of their
structure and functioning. Carbohydrates arc the major fuel source since about half
of all energy requirements of human arc satisfied from the oxidation of carbohydrates.
The leading role in fuel metabolism belongs to glucose and glycogen. Carbohydrates
arc a part of structural and functional components of cells. They include pentoses
of nucleotides and nucleic acids, carbohydrates of glycolipids and glycoproteins,
hetcropolysaccharides of the extracellular matrix. A large number of proteins, such
as enzymes, transport proteins, receptors and hormones arc glycoproteins in. which
carbohydrate components increase protein specificity. Lipids and some amino
acids can be synthesized from carbohydrates. Thus, carbohydrates perform diverse
functions, and each of them is vital lor an organism. The use of carbohydrates as an
energy source has great significance for an organism.
The major dietary carbohydrates arc starch, sucrose, lactose and indigestible fiber
(Fig. 6.1). Starch is the storage form of carbohydrates in plants. It is composed of
amylose containing unbranched chains with glucose units linked by ort I -4)-glycosidic
bonds and amylopectin, the highly branched molecule with a( l-4)-linkcd linear
glucose chains and a(. I-6blinked branch points. Sucrose, a component of dietary
sugar and fruits, contains glucose linked by fl-lt2 bond to fructose. Lactose, milk,
sugar, contains galactose linked by R-L4 bond to glucose. Starch and sucrose arc
predominant in the adult’s diet, lactose is the main carbohydrate in the child s diet.
254 Chapter 6. Carbohydrate metabolism

ai/si
l+CCli, > II C— D
/I
IIO
■OH— C“I4
CH/MH
II—C— CHI
14
fl—c—OH
[ frucinno'.
CH/JII

Glkssc tSHLtKC fructose

iuCMTKT i saner tamer

|STARCH |

■ Glucono

Efcflo 1,4 Linkages

Fig. 6.1. Structures of monosaccharides, ^saccharides. polysaccharides

Glycogen is the storage polysaccharide in animals and is a more highly branched

structure than amylopectin. Inulin is a polysaccharide of fructose. It is found in tubers
and roots of dahlias, artichokes, and dandelions. It is readily soluble in water and is
used to determine the glomerular filtration rale. Inulin is not hydrolyzed by intestinal
enzymes. Dextrins are intermediates in the hydrolyzis of starch. Cellulose is the chief
constituent of plant cell walls. It is insoluble and is not digested in mammals because
of lack any enzyme that hydrolyzes the [i (I -4) bonds.
Cellulose is an important source of 4nilk* in the diet and is a major component of
dietary fiber.
6.1. Digestion, Absorption and Transport of Dietary Carbohydrates. Disorders of Digestion... 255

Dietary carbohydrates arc cleaved in a digestive tract to monomers by glycosidases

which catalyze the hydrolyzis of giycosidic bonds (Fig. 6.2). Digestion of starch
begins in. a month. Salivary glands synthesize the amylase, a(l-4) glycosidase- which
catalyzes the hydrolytic cleavage of ct(l—4) giycosidic bonds. Starch is digested in a
mouth only partially since food is not there for a long time. A small intestine is the
main place of starch digestion where amylase as a part of pancreas juice is secreted.
The amylase splits giycosidic bonds anywhere and doesn’t hydrolyze disaccharides.
Oligosaccharides and maltose arc formed as a result. An isomaltosc is formed of those
glucose residues which are connected by a(l-6) giycosidic bonds in a molecule of
starch. Sucrose and lactose arc ingested with food.

Fig. 62. Steps of digestion of dietary cardo hydrates

Oligosaccharides and disaccharides arc hydrolyzed by specific glycosidases of a

small intestine. These enzymes produced by intestinal epithelial cells arc not secreted.
They arc located in the brush border membrane of absorptive cells in the intestinal
villi forming the big complexes with different substrate specificity. The sucra.se-
isomaltasc complex hydrolyzes maltose, isomaltosc and sucrose. The complex is
composed of two enzyme subunits. Both enzyme subunits have maltase activity and
hydrolyze a(l-4) glycosyl bonds of maltose releasing two glucose residues. The
sucrase subunit converts sucrose to glucose and fructose. The isomaltase subunit
cleaves a(I-6)—linkages of isomaltosc releasing glucose residues. Glucoamylase
complex containing two enzyme subunits cleaves the a(l-4) bonds between glycosyl
units in oligosaccharides and maltoses and releases glucose residues. p-Giycosidase
complex (lactase) converts lactose to glucose and galactose. Trchalase hydrolyzes the
256 Chapter 6. Carbohydrate metabolism

a(l—I) glycosidic bonds in trehalose, a sugar found in insects, algae, mushrooms.

These complexes of enzymes convert di saccharides, trisaccharidcs and small
oligosaccharides to monosaccharides, glucose, galactose and fructose, which enter
the blood through cells of the intestine.
The predominant monosaccharide formed as a result of the digestion of
carbohydrates is glucose since starch is a glucose polymer, and dietary disaccharides
(sucrose and lactose) arc also half constructed of glucose. The glucose coming from
the intestinal lumen with the blood of the portal vein enters the liver where part of
it is retained, and pan enters the cells of other organs and tissues through the main
blood How. Consumption of glucose by cells occurs with the participation of special
transport proteins forming hydrophilic transmembrane channels. There are two main
mechanisms of glucose transport: active transport depending on the concentration
gradient of sodium ionsand facilitated diffusion (Fig. 6.3).

Fig. 6.3. Different transport mechanisms for iitDiiosaccharitfes

There are two main types of glucose receptors. Sodium-dependent receptors

are found only in the kidneys and intestines and provide the glucose reabsorption
from the renal tubules and absorption from the intestinal lumen against the
concentration gradient. Facilitated diffusion. (GLUT=glucose transporters)
receptors arc found in all tissues (Table 6.1). Na'-dependent facilitated transport
(cotransport) is an energy-requiring process. Na',K\-ATPase pumps Na*
out of the cell into the blood and maintains a low intracellular concentration
of Na' - Glucose moves from low glucose concentration in the lumen to higher
concentration within the cell and at the same time Na' is transported down a
concentration gradient from the lumen to the cell. Facilitated transport is
6.1. Digestion, Absorption and Transport of Dietary Carbohydrates. Disorders of Digestion... 257

mediated by facilitaiivc glucose transporters which arc located on the luminal

side of the absorptive cells (GLUT), in this case, glucose moves from a high
glucose concentration in the lumen to a lower glucose concentration within the
cell. The faciliiaiivc glucose transporters exist in different cells as a family of
similar proteins designated GLUT I to GLUT 5.
Table 6.1. Localtzation of me glucose transport proteins

GLUT 1 Human erythrocyte

Blood-brain barrier
Blood-retinal barrier
Blood-placental barrier
Blood-testrs barrier
Liver

GLUT 2 Kidney
Pancreatic-cell
Serosal surface oi intestinal
GLUT 3 Brain mucosa cells (neurons}
GLUT 4 Adipose tissue
Skeletal muscle
Heart muscle
GLUT 5 Intesti rial epitlie li um
Spermatozoa

These transportersexist in the membrane in twro conformation slates. Extracellular

glucose binds the transporter, which then alters its conformation, transporting glucose
across the cell membrane (Fig. 6.4).

Glucose
Glucose
/iransponer
E iciracejular (bssie 1|

Glucose
Jiran^ponef
Ejciraceiular

Fig. 6.4. Conformational states of glucose transporter

258 Chapter 6. Carbohydrate metabolism

All receptors can be found both in die plasma membrane of the celt and in the
membranes of die vesicle in die cytoplasm. The number of receptors L 2. 3 and 5 in
die plasma membrane is al most constant a nd does not depend on the insulin level. In
die absence of insulin, GLUT 4 is almost completely in the cytosolic vesicles. Insulin
stimulates the movement of vesicles to the plasma membrane and their fusion as
a result of which the receptors arc embedded in the plasma membrane (Fig. 6.5).
When insulin concentration is low receptors are returned to the cytosol.

Fig. 6.5. Stimulation by insulin of glucose transport into muscle and adipose cells

Different monosaccharides have different mechanisms of absorption. Galactose

and glucose arc transported into the mucosal cells by an active, energy-requiring
process that requires a concurrent uptake of sodium ions; the transport protein is
the sod iu m-de pendent glucose cotransporter I. Fructose uptake requires a sodium-
independent monosaccharide transporter (GLUT-5) for its absorption. All three
monosaccharides arc transported from the intestinal mucosal cell into the portal
circulation by another transporter, GLUT-2.
6.1. Digestion, Absorption and Transport of Dietary Carbohydrates. Disorders of Digestion... 259

Glucose and other monosaccharides participate in further transformations

in cells only in the form of phosphoric esters (Fig. 6.6). Phosphorylation of free
monosaccharides leads to the formation of more reactive compounds and therefore
can be considered as an activation reaction. Entering the cells of organs and tissues,
glucose immediately undergoes phosphorylation using ATP. Phosphorylation of
glucose is an almost irreversible reaction since it runs using a significant amount
of energy. The formation of glucose-6-phosphatc in the cell is a peculiar «trap* for
glucose, since the cell membrane is impermeable to phosphorylated glucose (there
arc no corresponding transport proteins). In addition, phosphorylation reduces the
concentration, of free glucose in the cytoplasm. As a result, favourable conditions arc
created for the facilitated diffusion of glucose into cells from the blood.

ATP ADP

Hexokinase

Glucose | Glucose 6-phosphate

Fig. 6.6. Glucose photsphorylatiort reaction

There arc four isoenzymes that phosphorylate glucose (designated I to IV). They
arc encoded by four different genes and exhibit different Michaelis constant (Km)
values for glucose. Most hexokinase s have a low Km for glucose, and readily take
up glucose from the blood when blood glucose concentration is low (in the fasting
state). The liver isoenzyme of hexokinase called glucokinase has a high Km. The
brain hexokinase isoenzyme has a particularly low Km for glucose. The hexokinase
isoenzyme of myocytes has a high affinity for glucose. It is half-saturated at about
0.1 mM.
Differences in Km of glucokinase from Km of hexokinase correspond to the
conditions of liver enzymes functioning. In the postabsorptivc state, the blood glucose
concentration is about 5 mMol/L. At this concentration, the glucokinase reaction rate
is about one-fifth of the maximum rate, that is, the enzyme docs not work at full
capacity. During digestion, as a targe amount of glucose enters the portal vein and
further into the liver, its concentration in. the hepatocytes may exceed 10 mMol/L.
Accordingly, the rate of glucokinase reaction increases and a significant portion of
the glucose is retained in the liver. This prevents the excessive increase in glucose
concentration in the peripheral blood during digestion. Thus, the liver effectively
re moves the great a mounts ofglucose delivered by the portal blood after a carbohydrate-
rich meal (in the fed state) minimized hyperglycemia during the absorptive state.
Pathology of carbohydrates digestion can be the result of both defects of specific
enzymes that lake part in hydrolyzis of carbohydrates in the intestine and defects of
transport of monosaccharides through the absorptive cells of the in test inc.
260 Chapter 6. Carbohydrate metabolism

Any defect in a specific disaccharidasc activity of the intestinal mucosa causes the
passage of undigested carbohydrate into the colon. Asa consequence of the presence
of these osmotically active molecules, water is drawn from the mucosa into the colon,
causing osmotic diarrhea.
Lactose intolerance can be either the result of a primary deficiency of lactase
production in the small intestine or it can be secondary to an injury of the intestinal
mucosa where lactase is normally produced. Lactose intolerance may be acquired
and temporary. Il occurs with many gastrointestinal diseases, with some infectious
diseases, or after a stomach resection. The most characteristic manifestation of lactose
deficiency is diarrhea after milk intake. Un hydrolyzed lactose enters the lower pans
of the small intestine where it is fermented by intestinal Hora with the formation of
gases (flatulence) and acids. The osmotic activity of acids and gases causes an influx
of a large amount of water to the intestine and diarrhea arises. Flatulence is the reason
for the intestinal colic. Lactose deficiency disappears after the treatment of the main
disease. Temporary insufficiency of lactase in infants is especially dangerous because
their main food is milk. If the failure is not recognized in lime, severe dystrophy can
occur. More than three-quarters of the world’s adults a re lactose intolerant. Treatment
for this disorder is to reduce consumption of milk while eating yogurts and cheeses,
as well as green, vegetables such as broccoli, to ensure adequate calcium intake; to use
lactase-treated products; or to take lactase in pill form before eating.

6.2. GLUCOSE CATABOLISM. AEROBIC GLYCOLYSIS, ANAEROBIC

GLYCOLYSIS AND COMPLETE OXIDATION OF GLUCOSE
Glycolysis is an oxidative specific pathway of glucose catabolism by which one
glucose molecule is enzymatically split into two molecules of pyruvate (aerobic
glycolysis. Fig. 6.7, reactions 1-10) or two molecules of lactate (anaerobic glycolysis.
Fig. 6.8, reactions I-11). Aerobic and anaerobic glycolysis begin with a glucose
phosphorylation reaction and the formation of glucosc-6-phosphale. All glycolytic
intermediates arc also in the phosphorylated form. The sources of phosphate groups
in the phosphorylation reactions arc ATP and H3PO4.
All stages of the glycolysis occur in the cytosol of all cells of the body. Most
glycolytic reactions with the exception of three (Fig. 6.9; reactions 1, 3, 10) are
reversible. The principal function of glycolysis is the generation of ATP. Glycolysis
also provides precursors for fatly acids biosynthesis and the synthesis of amino acids
and pentoses.
Anaerobic glycolysis is a process that functions in the absence of oxygen. The
final product of anaerobic glycolysis is lactate. In some cell types anaerobic glycolysis
generates all of the cell’s ATP requirements (in RBCs which lack mitochondria) or
al least a portion of the cell’s ATP requirements (in skeletal muscles at the onset of
exercise and during intensive exercise and in tissues like lymphocytes, white blood
cells, the kidney medulla). Aerobic glycolysis is a process that functions when oxygen
is available. The final product of aerobic glycolysis is pyruvate.
In aerobic and anaerobic glycolysis one molecule of glucose (a six-carbon
compound) is convened to fructose-1,6-^phosphate (also a six-carbon compound),
which eventually forms two molecules of pyruvate/lactate (a th rec-carbon compound).
6.2. Glucose Catabolism. Aerobic Glycolysis. Anaerobic Glycolysis and Complete Oxidation... 261

Pre-paraTWy pfia&e
Phosphwyiaiion ol gkicose
and iis conversion ra
pfirnng
glycerafclehyde
■3-phosphare

Glucose e-prssphaie
o Hexckinase

Phosph oftejcsse

Fjuoqsg c ptBSfhae 0 jsoiTjera&e

Ptwspno-
Second _. L- TruciDkiftase-l
priming (aJ I
reason K Aldolase

Triosephosphaie
O isomerase

SyccraBc^pic 3-pftxfftrtc
Com piste oxidation of glucose

OjT.iiic9cp:clo*K- phosphate

Gtycei'Bidehyde 3phosphaie f2| ATP syflihesis phase

ZPi
2KAD*

1,3-&sphospncgiycofae |2]-
Fira ATP-
tarnfc-g - 2 ADP
resown vj
(subsvae level *
phospnor yiairznj
aPhosptngiyGerae (2)

2-PtKKpmglycerae (2)

Phosptweno^yfuvaie |2)
Second ATP
- 2 ADJ’
L’ATP
IsdKiiaK-fewel
ptostfHaylateyij
Pyfuvaie |2)

t
Aca^-CoA

f
TCAGyde
COh, hUO, ATP

Fig. 67. (A) Aerobic glycolysis: (B) Complete oxidation of glucose

262 Chapter 6. Carbohydrate metabolism

Glucose

ATP

Glucose-O-phosphate

112
Fructose-6-phasphaie

ATP
I3
Fructose-1,6-bisphosphale

4 Dihydroxyacelorte phosphate

Glyceraldehyde-3-p hospha le

1,3-Bisphosphnglycerale

3-Phosphog tycorale

■II
2-Phosphog lycerale

9
II
Phosphoenolpyruvale

Pyruvate

MAD+

fig. G.& Anaerobic glycolysis. Enzymes: 1 — hejtokinase/glucokinase (in liver); 2 —

phospnoglucose isomerase; 3 — phospriofructokinase 1:4 — aldolase; 5 — triose phosphate
isomerase: 6 — glyceraldehyde 3-phosphate dehydrogeoase: 7 — phosphoglycerate kinase; 8 —
pnosphoglyceromutase; 9 — enolase; 10 — pyruvate kinase: 11 — lactate dehydrogenase
6.2. Glucose Catabolism. Aerobic Glycolysis. Anaerobic Glycolysis and Complete Oxidation... 263

Aerobic and anaerobic glycolytic pathways involve two stages. In the first stage
(Fig. 6.8. preparatory phase) conversion of glucose into fruetosc-l^-bisphosphaLc
(reactions 1-3) and then reversibly to two molecules of glyceraldehyde 3-phosphate
(reactions 4 and 5) proceeds with the consumption of two molecules of ATP lor each
molecule of glucose that is split.
in the second stage (ATP synthesis phase) the conversion of 2 moles of
glyceraldehyde 3-phosphate into 2 moles of pyruvate (steps: 6-10) is associated with
ATP formation. Al this stage, the dehydrogenation of glyceraldehyde-3-phosphate
and the formation of NADH + I I1 takes place (reaction 6).

FAD
Mitccnondrial

J KT
gly-D=r-3 3FDH

FADHj

Irawr mitochondrial Mitochondrial

membrane matrix

Fig. 6.9. Glycerol phosphate shuttle

In aerobic glycolysis regeneration of NAD' necessary lor the oxidation of new

glyceraldehyde-3-phosphate molecules is coupled to the electron transport chain.
Produced NADH cannot transfer hydrogen directly to the respiratory chain since the
mitochondrial membrane is impermeable to NADH. The transfer of hydrogen from
the cytosol into the mitochondria occurs with the participation of special mechanisms
called the shuttle systems. In this case, hydrogen is transported through the membrane
with the participation of pairs of substrates, one of which is oxidized in the cytosol and
the other in the mitochondria. Two separate methods arc known: the glycerophosphate
shuttle and malate aspanatc shuttle (Fig. 6.10, 6.11) which differ from each other by
hydrogen acceptors for ETC and consequently the amount of ATP synthesized.

Fig ELI 0. Mai ale as panale shuttle

264 Chapter 6. Carbohydrate metabolism

[n the glycerophosphate system hydrogen is transferred to the FAD-dependent

dehydrogenase, therefore P/O = 2.
The second system is energetically more efficient since hydrogen enters the ETC
through mitochondrial NAD* and the P/O ratio is 3.
In anaerobic glycolysis, NADH oxidation occurs as a result of reduction pyruvate
to lactate (Fig. 6.11) independently of ETC. The overall equilibrium of this reaction
strongly favours lactate formation.

NAD NADH + H

2H+ + 2e"

CHj—CH—
i z
OH
y
Lactate -dehydrogenase
------------------ S■ '
I
CHa—C—
o

2H* + 2e“ Pyruvate

NAD*" NADH+H

Fig. 6.11 Lactate dehytliogenase reaction

The enzyme lactate dehydrogenase (LOH) catalyses the conversion of lactate to

pyruvate in the presence of the coenzyme NAD4. LDH is composed of four subunits.
The subunits arc single polypeptide chains of either H (heart) or M (muscle) isoforms.
Hybridization of these chains gives rise to five possible isoenzymes per species. The
isozyme composition of different organs is not identical. For example, M4 isoenzyme
prevails in skeletal muscles, and H4 is found in cardiac muscle. Isozymes have a
different total charge of the molecule that allows to separate them by electrophoresis
and measure the activity (amount) of each of the enzymes. Lactate dehydrogenase
appears in the blood in a number of diseases. The lactate dehydrogenase test can be
used to detect tissue damage. A lactate dehydrogenase test allows determining where
and how much damage is taking place in the body because of the differentiation of
different types of lactate dehydrogenase. This method is used in clinical practice for
diagnosis.
Aerobic breakdown of glucose can occur in all organs and tissues. But many
organs use other sources of energy or other methods of ATP synthesis. The brain
is most dependent on the aerobic breakdown of glucose. It consumes about I00 g
of glucose per day. Therefore, both, a lack of glucose and hypoxia are manifested
primarily by symptoms that indicate a disturbance of brain function (dizziness, loss of
consciousness, convuls ions).
Anaerobic glycolysis of animals and humans can occur in many cell types, but
its significance for different organs varies. During prolonged physical activity, ATP
synthesis in muscles occurs mainly due to the aerobic breakdown of glucose. In
muscles, the intensity of this process is limited by the amount of oxygen entering the
6.2. Glucose Catabolism. Aerobic Glycolysis. Anaerobic Glycolysis and Complete Oxidation... 265

mitochondria and the activity of mitochondrial enzymes that ensure the complete
oxidation of glucose. Under these conditions, the anaerobic synthesis of ATP
increases dramatically and lactic acid accumulates in. muscles. After a night's sleep,
the concentration of lactate in the blood is 1-2 Mmol/L and after intense muscle
exercises, it can reach 20 Mmol/L. Anaerobic glycolysis is especially important for
short-term intensive work. For example, 20 m long run (for about 30 seconds) is fully
provided by anaerobic glycolysis. At the same time the rate of anaerobic glycolysis
decreases quite quickly and the rate of aerobic glycolysis increases. After 4-5 minutes
of running (distance is about 1-5 km), the energy is supplied equally by the aerobic
and anaerobic processes. Muscle activity at 10 km distance (about 30 minutes) is
almost completely provided by aerobic processes and during the first minute of
work anaerobic oxidation of glucose provides much more power than under further
muscular work. With longer muscle activity, more and more fatty acids arc used as
sources of energy rather than glucose.
Erythrocytes do not have mitochondria al all and their need for ATP arc met
completely by anaerobic glycolysis. Intensive glycolysis is also characteristic of
malignant tumour cells. This process is of less importance for the bean muscle, brain,
and kidneys.
Anaerobic conditions do not exist in livi ng tissues, so the term ^anaerobic* indicates
only that oxygen is not used in this process. Thus, the ratio of the share of aerobic
and anaerobic catabolism of glucose in energy production depends on the presence of
mitochondria in the cells, their number, as well as on the availability of oxygen.
Reactions associated with the synthesis of ATP occur in the second stage of
glycolysis. Anaerobic glycolysis is less energy efficient Lhan aerobic glycolysis.
ATP can be produced by reactions catalyzed by glyceraldehyde-3-phosphate
dehydrogenase (oxidative phosphorylation}. pyruvate kinase and phospboglyceratc
kinase (substrate-level phosphorylation) if cells have sufficiently high oxidative
capacity. Given the stoichiometric coefficient, the resulting value must be multiplied
by two. In the initial stages (reaction I and 3) 2 moles of ATP arc consumed. Thus,
aerobic glycolysis provides 6(8) moles of ATP per mole of glucose:

2*(2+2(3))-2 = 6(8} moles of ATP.

Wien glucose is oxidized completely to COT and H.,O, 36 or 38 moles of ATP arc
generated. In this case, pyruvate may enter mitochondria and be converted to acetyl -
CoA, which is oxidized by the TCA cycle generating additional ATP:

2 xThe energy yield from aerobic glycolysis + 2 X-Thc energy yield from the TCA cycle =
= 6 (8)+30=36 (38) moles of ATP.

Under anaerobic conditions (cells are limited by mitochondrial capacity or

oxygen availability) 4 moles of ATP are produced by reactions catalyzed by pyruvate
kinase (IO-th reaction) and phosphoglyceratc kinase (7-th reaction) (substrate-level
phosphorylation). Thus, the energy yield of anaerobic glycolysis is 2 moles of ATP per
mole of glucose.

2*(l+l)-2=2 moles of ATP

266 Chapter 6. Carbohydrate metabolism

Lactate, as the end-product of anaerobic glycolysis, is transported to other tissues

such as the liver, cardiac muscle where it turns into pyruvate, which can be then
oxidized in the TCA cycle to CO2 and H,0 to form .ATP. Oxidation of lactic acid in
the heart muscle not only leads to the formation of energy but also helps to maintain a
constant blood pH. The concentration of lactate in the blood depends on the intensity
and duration of the work. Under resting conditions, the lactate concentration js
I mMol/L: after hard work, it may exceed 15 mMol/L, which leads to a decrease in
blood pH (lacticacidosis).
Lactic acidosis can occur in a number of pathological states when the supply of
tissues with oxygen is disturbed. Under these conditions (c.g., myocardial infarction,
pulmonary embolism, bleeding) the energy needs of the cells arc satisfied by anaerobic
glycolysis which leads to an increase of lactate levels and a drop of pH below the
optimal level that is required lor enzymatic activities. The result of this can be a sharp
disturbance in cellular metabolism. The blood lactate level depends on the intensity
of lactate oxidation to CO2 and H O and its use as a substrate for glucose synthesis.
Consequently, the insufficient activity of enzymes and pyruvate dehydrogenase
complex catalyzing the conversion of pyruvate to acctyi-CoA as well as citrate cycle
enzymes and glucose synthesis enzymes from lactate can lead to the formation of an
excess of lactate and accordingly a decrease in the value of blood plasma pH.
ATP production in the glycolytic pathway is a primary energy source in muscles.
Therefore, the rate of ATP synthesis should correlate with energy consumption.
There arc three points of regulation within glycolysis and these points arc
hexokinase, phospho frac tokinasc and pyruvate kinase that catalyze irreversible
steps of the glycolytic pathway (Fig. 6.12). An indicator of ATP consumption is the
accumulation of ADP and AMP that arc products of the ATP breakdown. Even
a small consumption of ATP leads to a marked increase in ADP and AMP. The
ratio of ATP to ADP and AMP characterizes the energy status of the cell and these;
molecules serve as allosteric regulators of glucose oxidation. Phosphofructo kinase
is activated by AMP and is inhibited by ATP. AM P binding to the allosteric site of
phosphofructokinasc increases the affinity of the enzyme for fructose-6-phosphate
and increases the rate of its phosphorylation. An increase in the ATP levels relative
to the ADP concentrations reduces the rate of this reaction. Under these conditions
ATP and act as an inhibitor. Il binds to the allosteric site of the enzyme, causes
conformational changes and reduces its affinity for the fructo sc-6- phosphate.
A decrease in phosphofructokinase activity al the high ATP levels leads to the
accumulation of both fructosc-6-phosphate and glucose-6-phosphate and the latter
inhibits hexokinase. In many tissues (with the exception of the liver and p-cclls of the
pancreas) hexokinase is inhibited by glucose-6-phosphate. With the high ATP levels,
the rate of the TCA cycle and the respiratory chain decreases. Under these conditions
glycolysis also slows down. The regulation of enzymes of complete oxidation and the
respiratory chain is associated with a change in the con cent ration of NADH. ATP
and some metabolites. Thus, an increase in the NADH concentration inhibits some
allosteric enzymes of the TCA cycle (Pig. 6.12).
6.2. Glucose Catabolism. Aerobic Glycolysis. Anaerobic Glycolysis and Complete Oxidation... 2&7

ATP

s-1
Pnosphofruclokirta &e-1 1
Q AMP, F-2jB-6bP

9 ATP. citrate
Frucl.ose-1,6-bisP

I
Giyceri-jeiiyde-S P

NADH + H*

1.3-Ebphosphogtycerale

t - ATP
|
I
PEP
Pyruvate kinase
0 F-1.6 &SP
@ ATP
NAD* NADH

Lactate 2 Pyruvate

Fig. 6.12L Major sites of regulation in complete oxidation of glucose: 0 - activators: @ - i nnib itors

Review tests
I. Match the figure and the Idler.
CaLaboJic pathways of glucose:
1. Anaerobic glycolysis.
2. .Aerobic glycolysis.
3. Complete oxidation of glucose.
268 Chapter 6. Carbohydrate metabolism

The scheme of glucose catabolism:

Glucose

2Pyfuvare SLaoiaie

2Ace[yS-GoA

C '

2. Match the figure and the letter.

Glycolysis metabolites:
1. Phosphoenol pyruvate.
2. 3-phosphoglyceratc.
3. Glyceraldehyde 3-phosphalc.
Structural formulas of glycolysis metabolites:

d ®—O—Ctfe—

3. Match the figure and the letter.

Glycolytic reaction:
Participants:
A. NAD'.
B. ADP.
CNADH-H
D. HtPO4.
E. ATP.
Glycolytic reaction:

—a—ch,— Ort­ ® — O— CHn— CH­ © -O CHp— CH—

I
0"
on OU OH
6.2. Glucose Catabolism. Aerobic Glycolysis. Anaerobic Glycolysis and Complete Oxidation... 269

4. Match the figure and the letter.

Aerobic glycolysis:
Reaction of:
A. Dehydrogenation.
B. Substrate phosphorylation.
C. Dehydration.
D. Isomerisation.
E. Phosphorylation involving ATP.

Glucose
I
G iucasa-6-pnospnata
1

F rudose-O-pnosphate

EJ---- -1 Fructosa-1,5-Disphospnata

22' DiJiydroKyacetone phosphate

Gtyceratdebydo-S-pnosphate (2)

s— I
1.3-Bjsphospbogiycefa.te (2)

S----- 1
3-Phosprioglycefate (2)
I

2-Phospnogiycofate (2)
l
Phospnoeroipyruvato |2}
I

Pyruvate (2)

Situational Problems
I. A child was admitted to the clinic with diarrhea after feeding on milk. To
determine the diagnosis, a lactose tolerance test was conducted. The test
solution contained 50 grams of lactose and was taken on an empty stomach.
Blood glucose levels were measured regularly over the next two hours — 30,
60 and 90 min after test solution and it turned out that the blood glucose
concentration was increased slightly. Present the possible reasons for the results,
argue them. For this:
a) write a reaction of lactose digestion in the intestine, specify the enzyme:
b) explain why the blood glucose concentration was not increased;
c) indicate the molecular mechanisms of the monosaccharide transport to the
small intestine cells and answer whether the violation of their absorption can
lead to these symptoms.
270 Chapter 6. Carbohydrate metabolism

2. The patient’s blood glucose concentration was 160 mg/dL 30 minutes after
taking 100 g of sugar. The blood glucose level of the same patient 30 minutes
after eating 100 grams of bread was lower. Why? To answer
a) draw the structures of sucrose and starch:
bi describe lhe digestion of these carbohydrates, indicate the enzymes, the site
of their synthesis, and the parts of the gastrointestinal tract in which the
hydrolyzis of sucrose and starch occurs.
3. For breakfast, a student has a mu din with jam and milk. What carbohydrate
digestion productscan be absorbed into the blood? For answer:
a) name the polysaccharides and disaccharides that arc contained in the
student’s food, represent a scheme for their digestion, specify the enzymes;
b) describe the transport of digestion products from the intestinal cavity into the
cnicrocytcs at low and high concentrations in the intestinal cavity;
c) indicate the digestion product with an increased concentration in blood,
name its normal concentration, and its value an hour after the meal.
4. There is a sweet taste in the mouth with prolonged chewing food containing
starch. Why? For the answer:
a) list the main dietary carbohydrates:
b) name the enzymes which arc present in human saliva;
c) write the reaction scheme of carbohydrates digestion in the mouth.:
d) specify the products of digestion providing a sweet taste.
5. The sprinter finishes 100 m run: the stayer runs the tenth km. Point out the
differences in the energy supply of these runners. To solve lhe problem:
a) draw a diagram of glucose catabolism, which is an energy source for muscles
in the stayer:
b) indicate lhe energy yield of the glycolysis and the complete oxidation, of
glucose, the mechanisms of ATP synthesis;
c) write down the substrates involved in. the dehydrogenation reaction, specify
the path of hydrogen from one of the substrates to oxygen, in the ETC;
d) indicate the differences in lhe linal products and the energy yield of lhe
metabolic pathways providing ATP in the sprinter and stayer.
6. The lens of the eye is the light refracting medium of the eye, and it has no
mitochondria. Glucose is used as an energy source in the lens. Which metabolic
pathway of glucose supplies energy to the crystalline eye lens? To answer the
question:
a? write a diagram of the metabolic pathway that provides the eye’s lens with
ATP, specify enzymes and coenzymes;
b) mark the reactions associated with the ATP consumption and synthesis,
calculate the ATP yield during the process;
c) specify the mechanisms of ATP synthesis in the process:
d) name the tissues and cells in which the ATP synthesis is the same as in lhe
tens*
e) write the dehydrogenation reaction and the reaction of final product formation
occurring in lhe process;
6.3. Gluconeogenesis. Regulation of Glycolysis and Gluconeogenesis 271

D indicate the laic of the end product of the process and the consequences of i ts
accumulation.
7. The number of mitochondria and myoglobin in athletes’ skeletal muscles
increases. How will these people change the production of lactate in muscles
compared to untrained people with the same physical activity? For answer:
a) write the scheme of the metabolic pathway whose final product is lactate:
b) explain how and why the activity of the metabolic pathway differs in athletes
and untrained people.
8. If the food contains the predominant amount of peeled cereals or bread made
from high-grade (lour, H, hypovitaminosis may occur. What is the role of vitamin
B1 in carbohydrate metabolism in the body? For answer:
a) name the coenzyme that contains vitamin B( and enzymes requiring this
coenzyme to function;
b} write the pathway of the carbohydrates metabolism involving these enzymes,
and explain how the rate of the process changes under a lack of B j and why ;
c) indicate the reactions that include vitamin Br
9. Hypovitaminosis caused by Bs deficiency in diet occurs in many people in spring.
Drowsiness and increased fatigue arc the most characteristic symptoms of this
hypovitaminosis. Why can the B-deficiency lead to such states? For the answer:
a) specify the coenzyme derived from B5 and the metabolic pathway that
includes it:
b) draw the scheme of the metabolic pathway providing energy for muscle
activity and show reactions that include this coenzyme.

6.3. GLUCONEOGENESIS. REGULATION OF GLYCOLYSIS

AND GLUCONEOGENESIS
Gluconeogenesis. Gluconeogenesis is the .synthesis ofglucosc from noncarbohydratc
precursors. Glucose is a universal fuel for human cells and if blood glucose decreased
some tissues such as the brain, red blood cells, kidney medulla, lens and cornea of the
eye. testes, and exorcising muscle, that depend on glucose would suffer from a lack of
glucose as a metabolic fuel. Liver glycogen, an essential source of glucose, can meet
these needs for only 10-18 hours in the absence of dietary intake of carbohydrates.
During a prolonged fast, hepatic glycogen stores are depleted and glucose is formed
from noncarbohydrate precursors. The most important function of gluconeogenesis
is to maintain blood glucose levels during prolonged fasting and intensive physical
exercises. The process occurs mainly in the liver that is responsible for 85-95% of the
glucose production and less intensively in the kidney cortex, as well as in the intestinal
mucosa. These tissues contain glucose-6-phosphatase which catalyzes the free glucose
production.
272 Chapter 6. Carbohydrate metabolism

Carbon sources for gluconeogenesis depend on physiological states in humans.

Lactate produced by an exercising muscle and red blood and other tissues with a
low O, content serves as a source of carbon in gluconeogenesis during exercise. The
breakdown of adipose triacyiglyccrol during fasting and exercise releases glycerol
that serves as a source of carbon in gluconeogenesis. During starvation* the major
precursors for glucose formation are amino acids obtained by degradation of muscle
protein and connective tissue.
The formation of glucose docs not occur by a simple reversal of glycolysis because
the overall equilibrium of glycolysis strongly favours pyruvate formation. The most of
glycolytic and gluconeogenic reactions arc reversible and arc catalyzed by the same
enzymes. Gluconeogenesis and glycolysis differ at only three points (Fig. 6.13): the
conversion of pyruvate to phosphocnol pyruvate that occurs in two steps a nd is catalyzed
by two enzymes, instead of the single enzyme used for glycolysis, removing phosphate
from fructose 1,6-bisphosphatc to form fructose 6-phosphatc and removing phosphate
from glucose 6-phosphatc to form glucose. Thus glucose is not generated by reactions,
which arc simply reversals of glycolysis, and four reactions of gluconeogenesis arc
irreversible.
Gluconeogenesis requires both mitochondrial and cytosolic enzymes.
Pyruvate produced from lactate, alanine, and other amino acids is first converted
to oxaloacctatc by pyruvate carboxylase, a mitochondrial enzyme that requires
biotin and ATP.

COO’
CO2 ATP ADP 4-P,
CHa
!“ X
1
« J . Biotin
Cbfe
in rx
1
cocr pyruvate carboxylase COO"
(Pyruvate) fOxatoacarate ]

The oxaloacctatc formed from pyruvate must be reduced to malate by mitochondrial

malate dehydrogenase:

Oxaloacctatc + NADU + H‘ *—* Malate + NAD'

because the mitochondrial membrane has no transporter for oxaloacctatc.

6.3. Gluconeogenesis. Regulation of Glycolysis and Gluconeogenesis 273

ATP„ Glucose

adp -^As*

Fructose-6-P
ATP^
ADP ~'A'*
Frutfose-1,6-oisP

Giyceraicfenyde-S-P Dihydroxyacetone-P
NAD" NAD+ I

Glycerol
MALJH NADH
1 r3-bispliosphoglycerate
O
ADP 's>jiX’ ADP E
O
O
□
ATP ATP a
D
3-Pnosprogiycerale ■

I
2-Phosphoglycera.te

Fig. 6.13. Reactions of glycolysis and gluconeogenesis. Starting with pyruvate, most of the steps of
gluconeogenesis are simply reversals of those of glycolysis

Malate leaves the mitochondria for the cytosol through a passive antiport and
is then reconverted to oxa loace late. This reaction has two purposes: to provide an
important substrate for gluconeogenesis, and to provide OAA that can replenish the
TCA cycle intermediates that may become depleted.
274 Chapter 6. Carbohydrate metabolism

Oxaloacctaic is then decarboxylated and phosphorylated, forming

phosphocnol pyruvate by phosphocnolpyruvate carboxykinasc (PEPCK). a cytosolic
enzyme that requires GTP. The reaction is driven by the hydrolyzis of guanosine
triphosphate.

GTP CDs GDP CH2 O

I I
C—O— P—o-
P frosphoenolpyruvare
CJibuiykiMSe
coo-
[Phospnogrejlpyruvate

The combined actions of pyruvate carboxylase and PEP-carboxykinasc provide an

energetically favorable pathway from pyruvate to PEP.
Fructose-6-bisphosphatasc converts fructose L6-bisphosphatc to fructo®e-6-
phosphatc releasing inorganic phosphaLc:

Fructose 1,6-bisphosphate H,O * Fructose 6-phosphate Pi.

Glucose-6-phosphatase releases inorganic phosphate converting glucose

6-phosphate to free glucose, which enters the blood:

Glucose6-phosphate H,0-Glucose + Pi.

Gluconeogenesis precursors include- intermediates of glycolysis and the

tricarboxylic acid (TCA) cycle. Glycerol lactate, and the a-amino acids arc the most
important gluconeogenesis substrates (Fig. 6.14). Glycerol is released during the
hydrolyzis of triacyl glycerols in adipose tissue and is delivered by the blood to the
liver. Glycerol is phosphorylated by glycerol kinase to glycerol phosphate, which is
oxidized by glycerol phosphate dehydrogenase to dihydroxyacctonc phosphate. an
intermediate of glycolysis.

CH2OH CH2OH

CH-OH ATP ADP HO—C —H O NAD* NADH + H*~ c— 0 0

HO C H
< J. LrHj U ■ kJ
V. > CH2—O“ p—0-
Glycerol kinase [ Glycerol 3-phospitaie 1
CHjOH O" itehyxfrogeflese O"

(Glycerol: [Glycerol 3-phosphato; CShydroxy acetone

pnospnaie
6.3. Gluconeogenesis. Regulation of Glycolysis and Gluconeogenesis 275

Glucose Glycogen

j Glu-6-pnosphatase

Giucose-€-phosphate *------------- Glucose-1 -phosphate

I
F/uctoso-6-phosphate

I Fru-1.6-bisphosphatase

Fructose-!,6-Disphosphate

Malate
TCA cycle Iso citrate
l

Fumarate CO2

X Alpna-katogiutarate
Succinate
SuCCihyl-GoA
,/C C°2

Fig. 6.14. Gluconeogenesis from Ute most important gluconeogenesis substrates: glycerol. lactate, and
lite (x-amino acids

Amino acids derived from hydrolysis of tissue proteins arc the most important
sources of glucose during a fast. During the catabolism of many amino acids pyruvate
or oxaloacclatc arc formed as intermediate products and can be then involved into
the gluconeogenesis pathway. For example, alanine, the major gluconeogenic amino
acid, is converted toa-kctoacid pyruvate by alanine aminotransferase:
276 Chapter 6. Carbohydrate metabolism

CHg
Alanine
aminotransferase
H "C —NHg+

COO’ (i-KG Glutamate COO

Other a-kctoacids derived from the metabolism ofglucogenic ami no acids can enter
the TCA cycle and formoxaloacctate (QAA), a direct prccursorofphospho-enol pyruvate
(PEP). Amino acids and glycerol arc mainly used in glucose synthesis when fasting or
at a low carbohydrate diet. Under these conditions gluconeogenesis provides glucose
for the brain, while other organs obtain energy from the fatty acid oxidation.
Lactate is released into the blood by exercising skeletal muscle, and by cells that
lack mitochondria, such as red blood cells. This lactate is taken up by the liver and
reconverted to glucose, which is released back into the circulation. The physiological
role of gluconeogenesis from lactate is significantly different. Lactate formed in
anaerobic glycolysis is not the end product of metabolism, hut its formation is a dead­
end metabolic pathway. The only way to use lactic acid is to convert it back to pyruvate
by lactate dehydrogenase reaction:

Lactate 4- NAD*" pyruvate + NADH + H .

The lactate as the source of the pyruvate is important both during usual human
activity and starvation. Conversion of lactate to pyruvate is the first stage of lactate
utilization. The lactate formed in intensively working muscles or in cells with
prevail i ng anaerobic catabolism of glucose enters the blood and then is mainly trapped
by the liver, where it is converted to pyruvate. In the liver. NADH/NAD+ ratio is
lower than in contracting muscle, therefore, lactate dehydrogenase reaction proceeds
reverse, i.c. , in the direction of pyruvate formation from lactate. Pyruvate is partially
convened to glucose in gluconeogenesis, and newly synthesized glucose enters the
blood and is taken by skeletal muscles (the Cory cycle or the glucose-lactate cycle) and
is partially oxidized in the liver to CO2 and H 2O (energy of its oxidation can be used for
ATP synthesis, needed forgluconcogenctic reactions). In the muscles, transaminase
transforms a part of pyruvate to alanine, which is transported to the liver and there
again forms pyruvate (glucose-alanine cycle) (Fig. 6.15).
Cory cycle allows the effective functioning of many extrahepatic cells at the expense
of the liver and partly of the renal cortex, c.g.:
► the lack of mitochondria makes RBCs completely dependent on anaerobic
glycolysis for ATP production. Then the lactate is partly disposed of by the liver
and renal cortex:
► skeletal muscle cell sand particularly fast-twitch fibers contracting under low
oxygen conditions, such as during intense exercise, produce much lactate. This
could lead to an intracellular accumulation of lactate, and a consequent reduction
in intracellular pH. In the liver, a large part of the muscle lactate is convened to
glucose, thereby allowing the muscle to use ATP for the contraction.
6.3. Gluconeogenesis. Regulation of Glycolysis and Gluconeogenesis 277

Gluco&e

AJ arire

I * I

Fig. 6.15. The Cori cycle and giucase-alanine cycle. Tne cycling of lactate and glucose between
p eripne rai tissues and liver is called tne Cori cycle and includes tne fol low ing steps: tne flow of lactate
from me contracting muscle and erytnrocytes witn tne blood flow to me liver; glucose synthesis from
lactate in tne liver; tne flow of glucose from tne liver tn rough tne bloodstream to me working muscle
and to the red mood cells; use of glucose as an energy substrate Dy tne contracting muscle and red
blood cells with tne formation of lactate. Alanine plays a special role in transporting amino groups to
tne liver via a pathway called tne glucose-aianine cycle

Thus, the Cori cycle performs two important functions: supplies lactate utilization
and prevents lactate accumulation and pH decrease (lactic acidosis).
During gluconeogenesis three enzymes require high-energy phosphate bonds:
> pyruvate carboxylase (I mole of ATP);
> phosphocnolpyruvate carboxykinase < I mole of GT P);
> phosphoglycerate kinase (1 mole of ATP).
As 2 moles of pyruvate are required for the synthesis of I mole of glucose, a
total of 6 high energy phosphate bonds arc cleaved (4 moles of ATP and 2 moles of
GTP). Thus, three ATP molecules arc consumed for each molecule of lactate duri ng
gluconeogenesis since two molecules of lactate arc necessary for the glucose formation.
The total equation of gluconeogenesis is described as follows:

2 Lactate + 6 ATP +■ 6 H,0 -*Glucose + 6 ADP + 2 H2O.

The produced glucose can again enter the muscles, and there turn into lactic acid.
The total glycolysis reaction is described as follows:

Glucose + 2 ADP + 2 H,PO4- 2 Lactate + 2 ADP + 2H2O.

These two equations give a result of the Cori cycle. The working muscles gel 2ATP
by splitting 6 ATP in the liver. All glucose present in the body, both from diet and
synthesized, is ultimately aerobically oxidized to carbon dioxide and water. Anaerobic
oxidation serves as an auxiliary pathway of energy production from glucose. It has a
local (e.g., in red blood cells) or situational (in a working muscle) significance. The
product of anaerobic glycolysis, lactic acid, is also eventually oxidized by an aerobic
pathway. In adults, about SO g of glucose can be synthesized per day, mainly in the
liver and also in the cortex and intestinal mucosa. The biological significance of
gluconeogenesis is not only to return lactate to the metabolic pool of carbohydrates,
but also Lo provide the brain with glucose under a low carbohydrate diet and starvation.
278 Chapter 6. Carbohydrate metabolism

A decrease of lactate use as a substrate in the glucose synthesis caused by a defect

of gluco ncogeneiic enzyme can lead to the blood lactic concentration to increase and
pH to lower and, consequently, lactic acidosis. Short-term lactic acidosis is quite
common even in healthy people during intense, heavy-resistance exercise, which is
compensated by hyperventilation of the lungs and accelerated elimination of CO2. In
this case, H' reacts with HCO to form carbonic acid H,COr followed byconversion to
CO, and H3O. Under uncompensated lactic acidosis the blood lactate level increases
to 5 mmol/1 (normally up to 2 mmol/l)t the blood pH can be 7.25 or less (normal
736-7.44). The blood lactate increase can be caused by a disturbance of the pyruvate
metabolism stemming from:
► tissue hypoxia of different origin, causing the activation of anaerobic glycolysis:
► liver damage (toxic dystrophy, cirrhosis, etc.), which lead to the level of utilization
of lactate:
► hereditary defects of gluconeogenesis enzymes (in particular, in case ofglucosc-
6-phosphatase deficiency), leading to a disorder of the lactate use;
► defects of enzymes disrupt i ng the action of the pyruvate dehydrogenase complex
(PDH);
► hypovitaminosis B,, B,, PP (the coenzyme function is impaired).
Regulation of Glycolysis and Gluconeogenesis. Two opposite processes, glycolysis
and gluconeogenesis, can occur in the liver. The switching of liver metabolism from
glycolysis to gluconeogenesis and vice versa is regulated by:
► the availability of substrates:
► allosteric mechanisms regulating the activity of key enzymes (enzymes of
substrate cycles):
► covalent modification (phosphorylation, de phosphorylation) of key enzymes
promoting by insulin and glucagon;
► induction (repression) of the key enzymes synthesis.
The regulation of glycolysis and gluconeogenesis in the liver is directed at the
irreversible steps of glycolysis and gluconeogenesis, which form three substrate cycles.
The name ^substrate cyclo means the combination of reactions of substrate synthesis
and its degradation. Each of the irreversible glycolysis reactions, together with
corresponding gluconeogenesis reactions form, so-called substrate cycles (Fig. 6.16).
These cycles serve as the control points for the regulatory mechanisms application,
which direct glucose metabolism on the path of either glycolysis or gluconeogenesis.
If continuous conversion of substrates to products occurred (see I, 2 and 3 cycles)
energy would be consumed and no useful result would be produced. Regulatory
mechanisms (hormones, allosteric activators and inhibitors) prevent the occurrence
of such potential futile cycles.
The first substrate cycle is mainly regulated by glucose concentration. In fed state
(absorptive state), the blood glucose concentration increases (up to 1.20-140 mg/dL,
or 7-8 mmol/1), glucokinase activity becomes maximal and glucosc-6-phosphate
reaction is accelerated. Since liver glucokinase is not inhibited by glucosc-6-phosphate
(unlike a. muscle hexokinase), glucose-6-phosphate is mainly oxidized in glycolysis
and stored as glycogen.
6.3. Gluconeogenesis. Regulation of Glycolysis and Gluconeogenesis 279

After a cartxjhydrale meat During Fasting

Fig. 6J6. The three cycles called substrate cycles <potential futile cycles) are designated as 1.2 aid 3

The direction of carbon How of the second substrate cycle (Fig. 6.16, 2)
depends on the activity of phosphofructokinasc andl fructose-1 r6-bisphosphatc
phosphatase. After a high carbohydrate meal, an increase of the blood insulin
levels and a decrease of the blood glucagon, levels (i.c., an elevation of the blood
insulin/glucagon ratio) rise the concentration of fructose 2,6-bisphosphate. This
compound is not an intermediate of the glycolytic pathway, but a special allosteric
activator of phosphofrucloki.nasc-1 (PFK-1, the regulatory glycolytic enzyme)
and allosteric inhibitor of fructose-L6-bisphosphale phosphatase (the regulatory
gluconeogenic enzyme. Fructose-2,6-bisphosphatc is produced in tissues due to
phosphorylation of fruciosc-6-phosphate by the enzyme phosphofructokinasc-2/
fructose 2,6-bi.sphosphatase that has dual functions (i.c., it is bifunctional —
BFE, Fig 6.1.7)..
280 Chapter 6. Carbohydrate metabolism

f irtsufini'gjijcagon after high cartohydrale meal

l Insulin./glucagon during fasting

fig. 6.17. Reaction catalyzed Dy bifunctional enzyme. ;t acts as a kinase after a meal when me insulin/
glucagon ratio is elevated and as a phosphatase during fasting when the insulinrtjlucagon ratio is low.
The activity of the enzyme is altered by phosphorylation and depnospnorylation. It is phosphorylated
Dy protein kinase A in die fasting state ^when it acts as a phosphatase} and depnospnoryiated in tnb
fed state i wtien it acts as kinase)

The activity of BFE is associated with the nutrition rhythm and is regulated by
hormones. Wien the instilin/glucagon ratio is high (after a meal, absorptive state),
insulin activates phosphop rotci n phosphatase, and the enzyme is dcphosphorylatcd
(BFE-OH); its phosphofrtictokin;isc-2 activity is enhanced, and it synthesizes
fructose 2,6-bisphosphalc from fructose 6-phosphalc and ATP. The levels of fructose
2,6-bisphosphalc arc elevated, PFK-l is activated and glycolysis is stimulated.
When the insulin/glucagon ratio is low (during fasting, postab sorptive state),
glucagon activates the adenylate cyclase system and causes the activation of protein
kinase A and the enzyme becomes phosphorylated (BFE-OPOsHn). Phosphorylation
enhances the phosphatase activity and inhibits the kinase activity of the bifunctional
enzyme, and fructose 2,6-bisphosphalc is converted back to fructose 6-phosphatc.
This reaction is not a simple reversal olThe reaction by which fructose 2,6-bisphosphatc.
is synthesized. Synthesis utilizes ATP, but the conversion of true lose 2,6-bisphosphatc
to fructose 6-ph.osphaLe produces inorganic phosphate (Pi.) rather than ATP. When
fructose 2,6-bisphosphatc levels arc low, the rate of glycolysis is decreased because
PFK-l is not activated: it leads to glycolysis slowdown and a switch of liver metabolism
to gluconeogenesis.
Fructose 2,6-bisphosphalc regulates both glycolysis and gluconeogenesis only in
liver.
The third substrate cycle is mainly regulated by pyruvate kinase, which is inactive
in phosphorylated form and active when dcphosphorylatcd (Fig. 6.16, 3). In the fed
state, insulin activates phosphoprotcin phosphatase, which d'ephosphorylaics and
6.3. Gluconeogenesis. Regulation of Glycolysis and Gluconeogenesis 281

activates pyruvate kinase. Consequently, the conversion of phosphocnol pyruvate

io pyruvate is accelerated in the fed state. In the well-fed state, glucagon enhances
phosphorylation of pyruvate kinase via cyclic AMP and the cAM P-dependent protein
kinase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased
recycling of phosphoenol pyruvate to pyruvate and enhanced glucose synthesis.
The conversion of pyruvate to oxaloacctatc is catalyzed by a biotin-dependent
enzyme — pyruvate carboxylase with, the participation of ATP as an energy source.
Acetyl-CoA allostcrica.il y regulates this reaction. During fasting, the body begins to
use fatly acids as fuel molecules and acetyl-CoA as a product of fatly acid oxidation
activates pyruvate carboxylase and directs pyruvate to gluconeogenesis.
The regulation of substrate cycles I and 3 is coordinated by fructose-1,6-
bisphosphate, which is an allosteric activator of pyruvate kinase, in the fed state,
fructose-1T6-bisphosphatc concentration increases due to the acceleration of the
initial steps of glycolysis, that leads to additional activation of pyruvate kinase.
One of the major functions of glycolysis is the ATP generation, so the pathway
is regulated to maintain ATP homeostasis in all celts. ATP. A DP, AMP, as well as
NADH, NAD' serve as allosteric effectors of the key enzymes of glycolysis and
gluconeogenesis (Fig. 6.18).

Fig. 6.18. Regulation of gly colysis and gluconeogenesis in tne liver

At high ATP and NADH concentrations typical for the high-energy status of the
cell, the key enzymes of glycolysis, phosphofruclokinase, and pyruvate kinase, arc
inhibited causing inhibition of glycolysis. A high concentration of AMP activates
glycolytic enzyme phosphofruclokinase and inhibits gluconcogenctic enzyme
fructose-1,6-bi.sphosphate phosphatase, and ADP inhibits pyruvate carboxylase
stowing down gluconeogenesis. Thus, at the low energy status of the cell, glycolysis is
activated and gluconeogenesis is inhibited.
282 Chapter 6. Carbohydrate metabolism

Induction and repression of the cellular con cent ration of key enzymes is regulated
by hormones. Steroid hormones binding to intracellular receptors directly regulate
gene expression, increasing or decreasing the synthesis of key enzymes. Glucagon
and insulin signal transduction also affect the synthesis of key enzymes. But these
hormones change the activity of transcription factors that vary the synthesis of
glycolytic and gluconcogenctic regulatory enzymes. In the fed state, insulin induces
the synthesis of glucokinase, phospho fructokinasc and pyruvate kinase, which
leads to the activation of glycolysis and causes repression of phosphoenolpyruvate
carboxykinase and reduces the rate of gluconeogenesis. In the well-led state,
glucagon increases gene transcription and synthesis of key gluconcogenctic
enzymes, phosphoenol pyruvate carboxy kinase, fructose-1,6-bisphosphatasc, and
glucose-6-phosphatase, and gluconeogenesis is activated, in prolonged starvation,
steroid hormone cortisol causing the induction of the enzyme gluconeogenesis,
phosphocnolpyruvate carboxykinase, is of particular importance in stimulating
gluconeogenesis.

6.4. PENTOSE PHOSPHATE PATHWAY

The pentose phosphate pathway (PPP) is an alternative pathway of carbohydrate
metabolism.
No ATP is directly consumed or produced in the cycle. This process supplies
the cells with the coenzyme NAD PH (used as a hydrogen donor in reduction and
hydroxylation reactions) and provides the cells with ribose-5-phosphate (which is
involved in the synthesis of nucleotides and nucleic acids). The PPP occurs in the
cytosol of the cell. It includes two phases: irreversible oxidative reactions, followed by
a scries of reversible nonoxidativc intcrconversions.
The oxidative phase supplies cells with two main products NADPH + H and
pentose:

Glucose 6-phosphalc + 2 NA DP' + H,0 -*■

ribose-5-phosphate + 2 NADPH + 2H4 + CO2.

Pentose formation involves two dehydrogenation reactions. The coenzyme of

dehydrogenase is NADP". which is reduced to NADPH + H'. Pentoses arc formed as
a result of the oxidative decarboxylation reaction (Pig. 6.19).
Glucose: 6-phosphate dehydrogenase catalyzes an irreversible oxidation of glucose
6-phosphate to 6-phosphogluconolactonc in a reaction that is specific for NADP as
its coenzyme. The pentose phosphate pathway is regulated primarily at the glucose
6-phosphalc dehydrogenase reaction. NADPH is a potent competitive inhibitor of
the enzyme, and under most metabolic conditions, the ratio of NADPH/NADP’
is sufficiently high to substantially inhibit enzyme activity. Insulin uprcgulatcs
expression of the gene for glucose 6-phosphate dehydrogenase, and flux through the
pathway increases in the well-fed state. 6-Phosphogluconolactonc is hydrolyzed by
6-phosphogluconolactonc hydrolase. The reaction is irreversible and not rate-limiting.
The oxidative decarboxylation of the product, 6-phosphogluconate is catalyzed by
6-phosphogtuconaic dehydrogenase. This irreversible reaction produces a pentose
sugar-phosphate (ribulose 5-phosphalc), CO and the second molecule of NADPH.
6.4. Pentose Phosphate Pathway 283

HOCH

HUGH
3 -zcse 6 phosphate
_______

ctfcbpog-
' s' NADP+
Glucose &-phosphaie rMl-s^
dehyrtio^enase ! l -
NADPH + H+

16- Phoapho -glucono-d-iacEoneJ

HOCH
I
HCOH
H-nH [6- Phospho-gluconate]
I
CHgOPOf"
NADP*
5--yiuCC'I Iai -?
B^hospnogfuconaie .
dehydrogenase ■- naDPH + H+
J* aw
CHsOH
i-o

rtCOH ____________
I-iqq^ p-RitjujosB 5 phosphate'

CHaOPOl"

Phospfliopefiiose |
isomerase I

GHO

HCOH [ D-Ribose 5-phosphate ]

neo!
HC0h
CHaQPOi-

Fig. 6.19. Oxidative reactions of the pentose phosphate pathway. The end products are ribose
5-phosphate. CO.. and NADPH
284 Chapter 6. Carbohydrate metabolism

[n the nonoxidativc phase, the pathway catalyzes the interconversion of three-,

four-, five-, six-, and seven-carbon sugars in a series of nonoxidativc reactions that
can result in the synthesis of five-carbon sugars for nucleotide biosynthesis or the
conversion of excess five-carbon sugars into intermediates of the glycolytic pathway.
This stage involves reversible reactions of transfer of two and three carbon fragments
from one molecule to another (Fig. 6.18). All these reactions take place in the cytosol.
The enzymes, pentose phosphate isomerase, transketolase and transaldolasc are
involved in these transformations. In the nonoxidativc stage, only pentoses arc not
producing NADPH.

Oxidative reactions of
pentose phosphate pathway

' Ribose 5-ptiosphate r,c_: [Frucios-e 6-phosphate ■

ks/

■"W"
Epimerase
I

Xylulose S-phosphate] (Erythrose 4-phosphatej , Fruciose 6-pfiosphale

4C

Fruao&e 1,&■
bte^ho^aase
AMoiase
Triose phospnane
isixnerase
[ Xylulose S- phosphate J i G.yceraldehyde 3 phosphate 1

Fig. 6.20. Oxidative and nonoxitiative stages of Pentose Phosphate painway. The non oxi dative
reactions of the pentose phosphate pathway occur in all cell types synthesizing nucleotides
and nucleic acids. These reactions catalyze the interconversion of sugars containing three
to seven carbons. These reversible reactions permit ribulose 5-phosphate to be converted
either to ribose 5-phosphate (needed for nucleotide synthesis), or to intermediates of
glycolysis — fructose 6-phosphate and glyceraldehyde 3-phosphate

Transketolase uses coenzyme thiamine diphosphate (TPP), a diphosphoric ester

of vitamin Br The nonoxidativc stage of pentose formation is reversible; therefore, it
can serve to form hexoses from pentoses. Using this path, excess pentoses that exceed
the needs of the cell can be returned to the hexose pool. The pentose phosphate
pathway of glucose conversion, both oxidative and nonoxidativc, can function in
the liver, adipose tissue, breast, adrenal cortex, red blood cells, Lc., in organs where
hydroxylation and reduction reactions actively occur, for example, the synthesis of
fatly acids and cholesterol, and neutralization of xcnobiolics in the liver and reactive
oxygen species in R8Cs and other tissues.
6.4. Pentose Phosphate Pathway 285

Pentose phosphate cycle. The oxidative phase of PPP and the phase of the return of
pentoses to hexoses (nonoxidaiivc portion of PPP in the opposite direction) constitute
together a pentose-phosphate cycle in which one molecule of glucose is completely
broken down in one turn of the cycle. The pentose phosphate cycle functions primarily
in the liver and the adipose tissue (Fig. 6.18 13). The total equation of the pentose
phosphate cycle:

6 Glucose 6-P + 12 NADP+ + 2 HZO - 12(NADPH + H ) -* 5 Glucose 6-P + 6 COr

intermediate products of the pentose phosphate pathway, fructose-6-phosphatc,

glyceraldehyde 3-phosphate, can be included in aerobic and anaerobic oxidation and
serve as an energy source for ATP synthesis. The reactions of the PPP arc part of the
process of formation of hexoses from CO3 in plants during photosynthesis.
1 n red blood cells. N AD PH + H1 is used to protect these cells from being damaged
by free radicals, reactive oxygen species (ROS). Glutathione, a non-protcin tri peptide
composed of glutamic acid, cysteine, and glycine, is an antioxidant in RBCs.
Glutathione has two forms that it can exist in: the reduced glutathione (GSH) and
oxidized glutathione (GSSG)_ In the reduced state, the thiol group (-SH) of cysteine
is able to donate a reducing equivalent (FT+ c ) to other molecules, such as reactive
oxygen species, to neutralize them, c.g. to convert hydrogen peroxide into a water
molecule. I n this reaction., the reduced glutathione (G-SH) transforms to the oxidized
state G-S-S-G. When glutathione SH-groups interact with H,O2, cysteine residues in
hemoglobin protomers arc protected from oxidation by reactive oxygen species, and
therefore, conformation and function of Hb remain unchanged. Oxidized glutathione
GSSG docs not act as an antioxidant, and is transformed back into the useful form
of reduced glutathione. For regeneration of oxidized glutathione into the reduced
form, NADPH -l- H' is used as a hydrogen donor, which is formed in the reactions
of the oxidative phase of PPP, one of which is catalyzed by glucose-6-phosphatc
dehydrogenase (Fig. 6.19). The defect of glucose-6-phosphatc dehydrogenase in
RBCs leads to a deficiency of NADPH + H', a decrease in the concentration of the
reduced form of glutathione and the oxidation of SH-groups of hemoglobin molecules
with the formation of disulfide bonds. This process is accompanied by the aggregation
of hemoglobin subunits and the formation of Heinz bodies. Erythrocytes lose the
plasticity necessary for passage through the capillaries, the integrity of the membrane
is disturbed, which can lead to hemolysis.

Quantitative cnaraclenshcs:
1) the Diood lactate concentration al rest — 1 m Mol/l:
2) tne normal blood glucose concentiation is 80-100 mg/dL {3.3-5.5 mMoi f I);
3) tne energy yield of aerobic oxidation of glucose to CO3 and h30 is 38 (36) moles of ATP per 1 met
of glucose;
4) energy yield of anaerobic oxidation of glucose is 2 mol of ATP per 1 mol of glucose.
286 Chapter 6. Carbohydrate metabolism

Review tests
1. Match lhe figure and the letter.
Enzymes:
1. Phosphocnol pyruvate earboxykinase.
2. Pyruvate carboxylase.
3. Glycerol phosphate dehydrogenase.
Glycolysis and Gluconeogenesis in the liver:

I----------- -El
! 1.3-BispnosphoQlycefate J

1
I...... ............ G3
3-Phosphoglycerate__ )

(
I
2-PnospnoQiycerate

I
■phbsphoefldi-pyhjvate >>---•
(
E
rp^qaeetatg]
*[ Pyfuvata ] CD
2. Match lhe figure and the lellcr.
Property of an enzyme:
1. It contains biotin as a cofactor.
2. It is hydrolase.
3. It is oxidorcducrase.
Glycolysis and Gluconeogenesis in the liver:
(thediagram is presented in Lest I)
3. Match lhe figure and (he Idler.
Regulation of gluconeogenic enzy mes. The synthesis of an enzyme is:
1. Repressed by insulin.
2. Inhibited by fructose-2,6-bisphosphate.
3. inhibited with a decrease in Lhe ratio of ADP/ATP.
Glycolysis and Gluconeogenesis in the liver:
(the diagram is presented in test 11.
6.4. Pentose Phosphate Pathway 237

4. Match the iieurc and the Idler.

Glycolytic reaction:
Participants:
A. Frudosc- lT6-bisphosphatc.
B. BFE-O-P.
C. Fructose-2,6-b is phosphate.
D. Fruclosc-6-phosphaic.
E. BFE - OH.

Situational Problems
1. A person, ate 200 g of carbohydrates and then did not cal anything fora day. What
process of carbon metabolism was stimulated in the liver 14 hours after the last
meal? For answer:
a) write a diagram of the process that speeds up in the liver after 14 hours after
eating:
b) indicate the regulatory reactions and their enzymes;
c) describe the mechanism of signal transduction by a hormone that regulates
this metabolic, pathway.
2. A well-trained athlete is running 5 km. What is the difference in glucose
metabolism in the liver and muscle by the end of the race? For the answer:
a) write a diagram of glucose metabolism in the liver in this case:;
b) draw a metabolic pathway of glucose in muscles;
c) name regulatory enzymes and describe the mechanism of their activation;
c) explain the significance of the metabolic pathways that occur in the liver and
muscle.
3. The ATP source for the glucose synthesis from lactate can be the conversion
of lactate to pyruvate k subsequent oxidation of it to carbon dioxide and water.
How many moles of lactate must be oxidized in the liver to CO2and H3O to
provide ATP' for the synthesis of 1 mole of glucose from lactate? For answer:
a) depict the diagrams of the lactate oxidation and gluconeogenesis from lactate;
b) indicate the reactions produced and consumed ATP;
c) name the hormones that regulate gluconeogenesis in the liver and explain
the mechanism hormonal signal transduction.
4. During prolonged fasting and intense exercise, the product of fat breakdown
in adipose tissue, glycerol, is one of the gluconeogenesis substrates. How can
glycerol be involved in gluconeogenesis? How many moles of glycerol and ATP
arc required to synthesize I mole of glucose? To answer:
288 Chapter 6. Carbohydrate metabolism

a) write a diagram for the glucose synthesis from glycerol and name the reactions
occurring with ATP consumption;
b) name the hormones that stimulate gluconeogenesis and describe the
mechanism of signal transduction bv these hormones;
c) explain the role of the bi functional enzyme in the regulation of gluconeoge­
nesis.
5. During prolonged fasting, amino acids derived from muscle proteins and
connective tissue become the main substrate for gluconeogenesis. How docs
glutamate enter the gluconeogenesis? For answer
a> write a reaction of the glutamate conversion into a-kctoglutarate, name the
enzyme, indicate the energy effect of the reaction;
b) draw a diagram for the a-kctoglutarate conversion to oxaloacctatc and
consequent including of OAA to gluconeogenesis, specify the reactions
associated with. ATP consumption;
c) name the hormones that accelerate gluconeogenesis during fasting and
regulatory enzymes of the process; and describe the mechanism of signal
transduction by these hormones.
6. Students carry out a laboratory experiment with avidin (a protein in. egg white)
having a very high affinity for biotin enzymes. Which enzyme of glucose
metabolism would be inhibited by the addition of avidin to a cell homogenate?
For answer;
a) draw a reaction that would be blocked by the addition of avidin to a cell
homogenate.;
b) write a diagram of the carbohydrate metabolism in which this reaction takes
place.
7. Students carry out a laboratory experiment of the oxidative conversion of glucose
to ribose 5-phosphate on muscle and liver homogenates. The first carbon of
glucose is rad ioact ivcly labeled. Will the labeled atom appear in pentose? Which
tissue; liver or muscle will show a higher rate of the process? To solve the problem:
a) depict reactions of the oxidative stage of the pentose phosphate pathway using
chemical formulas;
b) indicate the significance of this process for the cells and for the whole organism;
c) name the regulatory enzyme and mechanism of its regulation.
8. In a patient with anemia, the presence of Heinz bodies in the RBCs was the
result of the hemoglobin subunits aggregation due to the oxidation of -SH
groups of Hb cysteine residues with active oxygen forms and the formation of
disulfide bonds. What metabolic disorders in the RBC can be the cause of this
clinical case? To solve the problem:
a) indicate which reactions maintain cysteine residues in a reduced state;
b) name the coenzyme involved in this process, write a diagram of the process in.
which the reduced form of this coenzyme is formed:
c) indicate the enzyme which deficiency may lead to a lack of the reduced
coenzyme and be the cause of the clinical case described above.
9. A patient was admitted to the hospital with suspicion of malaria. After the
examination was complete and the diagnosis confirmed, the patient was
6.5. Synthesis ana Degradation of Glycogen 289

prescribed primaquine, an a nt im alarial drug. The drug caused a complication

and led to hemolysis of erythrocytes and a decrease in the total number
of circulating erythrocytes. The screening showed a glucose-6-phosphate
dehydrogenase defect. Explain why the enzyme deficiency led to red blood cell
hemolysis in the patient taking primaquine (the drug induces the formation of
free radicals in cells). To answer:
a) write a diagram of the process involving glucose -6-phosphate dehydrogenase;
b) indicate the coenzyme of the enzyme and the reaction that includes its
reduced coenzyme;
c) explain the cause of REC hemolysis in. reduced coenzyme deficiency.

6.5. SYNTHESIS AND DEGRADATION OF GLYCOGEN

Glycogen is the main reserve polysaccharide found in the most animal cells.
Glycogen is a highly branched homopolysaccharide whose monomeric unit is
glucose. Glucosyl residues arc connected in linear chains with a 1,4-glycosidic
bonds and at branch points with al,6-glvcosidic bonds (Fig. 6.21). The glycogen
molecule is more branched than the starch molecule: branching points occur every
8- 10 glucose residues. The branched structure of glycogen provides a large number of
non reducing ends (terminal monomers), which contributes to rapid degradation and
rapid synthesis of glycogen because enzymes can work on many different glycogen
branches simultaneously.

Fig. 621. Glycogen structure. Glycogen is composed of glucosyl un its linked Dy cxl ,4-giycosid'ic don ds
and al ,6-glycosidic honds. Tne tranches occur more frequently in tne center of me molecule, and
less frequently in tne periphery

Glycogen is mainly deposited in both the liver and skeletal muscle and is stored in
large cytosolic granules. Glycogen granules are poorly soluble in water a nd do not affect
290 Chapter 6. Carbohydrate metabolism

the osmotic pressure in the cell. Some enzymes involved in glycogen metabolism arc
also associated with granules, which facilitate the enzyme interaction with substrates.
Synthesis of glycogen. Glycogen is synthesized in the absorptive state (1-2 hours
after carbohydrate ingestion), mainly in the liver and in muscles. Because of muscle
greater mass, muscle contains about three to four times as much glycogen as does
liver. The biosynthetic pathway is an energy-requiring pathway: the attachment of one
monomer to the polysaccharide chain is associated with the consumption of ATP and
UTP. As i n glycolysis, glucose is phosphorylated to glucose 6-phosphate, catalyzed by
hexokinase in muscle and glucokinase in liver. Glucose 6-phosphatc is isomerized to
glucose 1 -phosphate by phosphogluco mutase. Then glucose I-phosphate reacts with
uridine triphosphate t UTP) to form the active nucleotide uridine diphosphate glucose
and pyrophosphate (Tig. 6.22), catalyzed by UDP-glucose pyrophosphorylasc.

Fig. 622. Formation of an activated form of glucose, UDP-glucose, synthesized from glucose
1-phosphate and UTP by UDP-glucose pyrophosphorylase

The formed UDP-glucose is an immediate substrate for glycogen synthase, which

adds the glucose residue from UDP-glucose to a primer (a preexisting glycogen
molecule containing 4—8 glucosyl residues) to form an a I.,4-glucose bond. Wien
the length of the synthesized chain reaches Jl-l.2 glucose residues, the branching
enzyme, glucosyl-1.4-1,6-transferase, cleaves a 6- to 8-residue piece and reattaches to
a glycogen core by an a l,6-bond, forming a side chain. Then the lengthening of the
chains and their branching arc repeated many limes. The result is a highly branched
molecule containing up to I million glucose residues (Fig. 6.23).
6.5. Synthesis ana Degradation of Glycogen 291

■Giyccgsri core

UDP-^uccse

Glycogen synttiorLC

OOOCOC OCCOO Syccgen rare

SuC'P-duccse

6uDP
f'

JEIUCCXj' ■Styccgen core

4:6-Transferase
{tM-anchnig enzyme;-

Qyccgm core

Glycogen synthase

Fig. 623. Glyc ogeri synmes is

Glycogen degradation. Glycogen degradation (mobilization) occurs in the

postabsorptivc state or during fast (between meals} and accelerates during physical
activity. In humans, a substantial portion of liver glycogen is degraded within some
hours after a meal and after 24 hours of fasting, the liver glycogen is almost totally
depleted. Muscle glycogen can be exhausted in less than an hour during vigorous
activity.
Glycogen is degraded by two enzymes: glycogen phosphorylase and the
debranching enzyme with two catalytic activities. Glycogen phosphorylase catalyzes
the rate-limiting step in glycogenolysis by promoting the sequential phosphorolytic
cleavage by inorganic phosphate of the a 1,4 linkages of glycogen to yield glucose
I-phosphate (Fig. 6.24).
292 Chapter 6. Carbohydrate metabolism

CXDOCXKodBcOCOO Glycogen core

I P'’*
t Glycogen pncsphoiylise
0 Glucose 1 pneepnaieiC' .

4^
cooffiooooo Glycogen core

4:4-Ti'3ftsrerase

ooafiooooo Glysigan core

a-1,6-Glucosidase

X. IGkicoseQ}

- OOOOOOOCOO Glycogen core

GJycogefl pfro&ptioiyiase

Degradation ccrr.ifjes

Fig. 624. Glycogen degradation

The terminal glucosyl residues from the outermost chains of the glycogen
molecule arc sequentially removed by glycogen phosphorylase until approximately
four glucose residues remain on cither side of 1,6 branch. Glycogen phosphorylase
cannot act on the glycosidic bonds of the four glucosyl residues closest to a branch
point. The debranching enzyme acts both as transferase and as an ahb-glucosidasc.
Transferase catalyzes the removal of a trisaccharide unit from one branch and adds
to the other by an a 1,4-glycosidic bond, exposing a single glucose residue joined by
ana 1,6-glycosidic linkage, a 1,6-Glucosidase hydrolyzes the a I, 6-glycosi die bond,
resulting in the release of a free glucose molecule. Glucose I-phosphate, the end
product of glycogen degradation, formed in the phosphorolytic cleavage of glycogen,
is then converted by phosphogluco mu Lase into glucose 6-phosphate to enter the
metabolic mainstream:

Glucose 1-phospha te**Glucose 6-phosphate.

The breakdown of glycogen to glucose-6-phosphale and free glucose occurs

without the ATP consumption.
Mobilization of glycogen in the liver differs from that in muscles by a single
reaction, due to the presence of the enzyme glucose-6-phosphatasc in the liver.

Glucose 6-phosphatc ■+ H3O glucose + Pr

6.6. Regulation of Glycogen Metabolism 293

Glucose 6-phosphatasc is absent from most other tissues. Liver glucose-6-

phosphatase provides the main function of liver glycogen to release glucose into
the blood when the blood glucose level drops, as it does between meals. Thus,
glucose is not a major fuel for the liver, and the mobilization of liver glycogen
ensures the maintenance of constant blood glucose level in the postabsorptivc state
(3.3—5_5 mmol/dL). After 10—18 hours after a meal, glycogen, stores in the liver are
significantly depleted, and fasting for 24 hours leads to its complete exhaustion. The
function of muscle glycogen is to provide cells with glucose-6-phosphatc, used in the
muscle itself for oxidation and for energy, or other purposes (Fig. 6.25).
Glycogen
Liver J Muscle
Glucose-1 -ptiospnats

Other pathways of G-6-P metabolism

Fig. G25. The pathways of glucose 6-phosphate

6.6. REGULATION OF GLYCOGEN METABOLISM

Glycogen metabolism is precisely controlled by multiple mechanisms, and the
focus of this control is regulatory enzymes. Switching of synthesis and degradation of
glycogen in the liver and muscles occurs with the changing physiological conditions
from the absorptive state to the poslabsorptive stale and from the rest to muscle
exercises. Insulin, glucagon and epinephrine arc involved in the regulation of these
metabolic pathways in the liver, and insulin and epinephrine control glycogen
metabolism in the muscles.
The rate-limiting steps of glycogen synthesis and glycogen degradation arc
catalysed by the enzymes, glycogen phosphorylase and glycogen synthase, regulated by
allosteric mechanisms and covalent modifications due to reversible phosphorylation
and de phosphorylation of enzyme protein in response to hormone action.
294 Chapter 6. Carbohydrate metabolism

Regulation of glycogen metabolism in the liver. The primary stimulus for insulin and
glucagon secretion is the changing blood glucose concentration. After carbohydrate
meal, the blood glucose level elevates to 10-12 mmol/L increasing insulin level and
decreasing glucagon level. Thus, iiisulin/glucagon ratio increases and insulin effect
becomes predominant. Insulin influences the following:
► acceleration of glucose transport into cells of insulin-dependent tissues (muscle
and adipose tissue);
► change in enzyme activity by phosphorylation and dcphosphorylation (Fig. 6.26).

Membrane

Protein phosphatase
inactive

Cascade of
phosphorylation
reaction

Protein phosphatase active

Glycogen synthase <Pi Glycogen phosphorylase

inactive active

Glycogen synthase - OH Glycogen phosphorylase - OH

active inactive

Fig. 626. insulin effect on glycogen synthase and glycogen phosphorylase

For example, insulin activates phosphodiesterase and reduces the concentration

ofcAMP in the cell. In addition, insulin activates phosphoprotcin phosphatase
of glycogen granules, which dephosphorylaics glycogen synthase and converts
it to the active state. Dcphosphorylation of glycogen phosphorylase under
the influence of phosphoprotcin phosphatase, on. the contrary, leads to its
inactivation:
► changing the amount of principle enzymes by induction and repression of their
synthesis. In the liver, insulin induces glucokinase synthesis, thereby accelerating
glucose phosphorylation.
Therefore, insulin, increases the rate of glycogen synthesis.
In the postabsorptive state, the low blood glucose concentration decreases the
insulin-glucagon ratio and glucagon stimulates the breakdown of the liver glycogen.
Glucagon ^triggers* the adenylate cyclase cascade of reactions, leading to the activation
of glycogen phosphorylase and inhibition of glycogen synthase (Fig. 6.27).
6.6. Regulation of Glycogen Metabolism 295

Glucagon (her ody)

i Glucose

AdOfiytllC Hiospiw
■EVdKC
fficsi erase?

Cyropiasfn
Glucose
i
’ GkKOftBUrU
'V
Praiein.
Fleguiaory Glucose 6-phosphate
kinase A
(riactreeji SiikHil-€AMP %
GlpZOQZF i
l
« AvxsptKqftaw- . &jrrtrusc-P •
r kmasc » Glucose 1 -phosphate
Ijnacimi'l Ki
Protein; ADP PfctDTl
3 i' Active protein <5 j
ptcs|.-^aLase phDfptnlafK'

w kinase A J
%
ATP .4

PhOtJpjKQkKC- ADP ■■ - *
' Sroftasc *•
HKKKC-P ■jnacb¥c$
nsdNc'l

Glycogen UDP-glucose *
Oycogcn
phcKphocyniw t:
ijrurtvc'i
K

Glucose 1-phosphan Glucose G^rtiosphaie

Rrarein
itfioGpfiaiase Live I Glucose
6-ptiosphaiase

Blood
dizxjse

Fig. 6^7. Regulation of glycogen synthesis anti degradation in me liver 1 — Glucagon and epinephrine
interact with specific membranereceptors. The hormone receptor complex activates adenylate cyclase
via G proteins which synthesizes CAMP from ATP. 2 — CAMP binds io P KA and activates the catalytic
subunits. 3 — PKA activates phosphorylase kinase by phosphorylation. 4 — Phosphorylase kinase
converts glycogen phosphorylase o to the active glycogen phosphorylase a by phosphorylation. 5 —
PKA phosphorylates glycogen synthase and decreases its activity. 6 — inhibition of glycogen synthase
and the activation of glycogen phosphorylase I eact to glycogen degradation to glucose 1 -phosphate

Epinephrine released from the adrenal medulla in response to neural signals

reflecting an increased demand for glucose stimulates glycogenolysis through two
different types of receptors: the ar and p-agonist receptors. Regulation of glycogen
degradation and synthesis in the liver by epinephrine acting at the p-receptors and
glucagon arc similar (Tig. 6.28). The epinephrine binding to a preceptors in die
hepatocyte activates glycogenolysis and inhibits glycogen synthesis principally by
increasing the Ca2 levels in the liver. The effects of epinephrine at the a1-receptor arc
mediated by the phosphatidylinositol bisphosphate (PIP2)-Ca21 signal transduction
system (Fig. 6.28).
296 Chapter 6. Carbohydrate metabolism

Epinenaluine

a-Agonis Ptwsj-MTcSpase C — Protein Jdnase C

receptor
■roecwrc uuuxw

XaXKODtt

Cytoplasm

GByGag&n
Calmodulin - synthase
(ama dive)
dependent
protein fanase
Glycogen synthase
(active)
Endoplasmic Ca1*- calmodUsn
reticulum Glycogen
II
■i phosphorylase a — p
W.
(active)
Kinase
o
Glycogen
ptiosplwylase t>
(inactive)

Fig. 6.28. Regulation of glycogen synthesis and degradation in the liver by epinephrine. 1
Epinephrine binds to <i .-receptors in the liver. Epinepnrine-receptor complex transmits a signal via G
proteins to pnospnoiipase C. Pnospnolipase C hydrolyzes PlP2 to DAG and IPS.2 — IPS stimulates tne
release of Ca?* from the endoplasmic reticulum. 3 — Ca3, binds to me protein calmodulin. Calmodulin-
dependent protein kinase and phosphorylase kinase are activated. Both Ca3, and DAG activate protein
kinase C. -4 — These three kinases phosphorylate glycogen synthase at different sites and decrease
its activity. 5 — Phosphorylase kinase phosphorylates glycogen phosphorylase to the active form
activating glycogenolysis and inhibiting glycogen synthesis

Thus, the epinephrine signal transduction stimulates the phosphorylation of key

enzymes, the change in their activity, and the switching of glycogen synthesis to
its breakdown through two different types of receptors: the af-receptors (adenylate
cyclase mechanism} and p-agonist receptors (inositol phosphate mechanism).
Regulation of glycogen metabolism in muscles. In the absorptive state, muscle
cells actively consume blood glucose and synthesize glycogen. This is facilitated by
alimentary’ hyperglycemia and by the GLUT-4 movement from the cytosol to the
plasma membrane that is stimulated insulin. Myocytes lack receptors forglucagon and
the breakdown of glycogen is mainly stimulated by Ca3' ions and epinephrine similar
to those occurring in the liver. However, the source of Ca2' in muscles is primarily
Ca2 release from the sarcoplasmic reticulum during neurostimulation, and not as a
result of the action epinephrine.
In the case of short-term muscular loads, the main energy source is glucose,
which enters the muscles from the blood (as glucose I-phosphate) and from the
muscle glycogen stored (Note that 100 g of glycogen can provide running for about
15 minutes).
6.6. Regulation of Glycogen Metabolism 297

At the transition from rest to vigorous muscular activity, the need) of skeletal
muscles for energy increases tenfold. In response to a signal from the central
nervous system epinephrine secreted from the adrenal medulla interacts with
receptors of muscle cell membranes and triggers the cascade mechanism (adenylate
cyclase, inositol phosphate, associated with calmodulin (Fig. 6,29, 2 and 3), which
provides fast activation of energy supplying reactions. Such cascades allow for large
amplification of the initial signal. For example, in the adenylate cyclase cascade, one
epinephrine molecule activates one adenylate cyclase molecule, then one molecule of
adenylate cyclase can synthesize many molecules ofcAMP and a signal is amplified.
In the same way, the signal is enhanced at all enzymatic stages. Thus, the cascade
mechanism ensures the inclusion of large amounts of glucose in catabolism in a short
time.

Epinephrine

Fig. 629. Activation of muscle glycogen phosphorylase by muscle contraction, neural impulses, ano
epinephrine. 1 - AMP produced from the degradation of ATP during muscular contraction aitostericaily
activates glycogen phosphorylase b. 2 — The neural impulses that initiate contraction release Ca3- from
the sarcoplasmic reticulum. The Cat, binds to calmodulin, wliich is a modifier protein mat activates
phospnorylase kinase. 3 — Phosphorylase kinase is also activated through phosphorylation by FKA.
The formation of CAMP ana the resultant activation of PKA are initiated toy the binding of epinephrine
to plasma membrane receptors

When the muscle returns to rest, epinephrine secretion, stops. Having been already
released epinephrine is destroyed and results in adenylate cyclase inactivation.
cAMP present in the cell is converted to AMP by phosphodiesterase, and therefore.
298 Chapter 6. Carbohydrate metabolism

protein kinases are inactivated; glycogen phosphorylase and glycogen synthetase arc
dcphosphorylatcd by phosphatases, and the system returns to a state where glycogen
mobilization is suppressed , but its synthesis can occur.
The cascade mechanism is triggered in muscles under intensive and severe muscle
activity. Under moderate load (mild muscle exercises), glycogen phosphorylase
practically remains in dcphosphorylatcd inactive state, hut the breakdown of
glycogen occurs nonetheless. This is due to the fact that phosphorylase-OH
can also be activated by covalent modification (Fig. 6.29, I). In the contracting
muscles, the AMP concentration increases due to reaction: 2ADPH - ATP
-I- AMP. AMP is an allosteric activator of muscle phosphorylase (AMP docs not
activate hepatic phosphorylase). Muscle phosphorylase is a homodi me r; each of
the protomers contains an AMP binding site and a phosphorylation site. Extremely
active phosphorylase is phosphorylated and is associated with AMP. Intermediate
phosphorylases arc less active and allow to perform moderate physical exercises.
For example, in experimental mutant mice with phosphorylase kinase deficiency,
the formation of phosphorylase-P is generally impossible. These mice do not dilTcr
from normal animals in normal motor activity (swimming in water for a long time}.
When frightened instead of rushing, such mice will have convulsions as a result of the
impossibility ofa rapid glycogen mobilization.
The significance of glycogen metabolism regulation. When a hormonal signal is
transmitted through intracellular mediators, its considerable amplification occurs:
therefore, activation ofglycogen phosphorylase via any signal transduction system in the
liver allows rapidly obtaining a large amount of glucose from glycogen. Strengthening
the hormonal signal in the muscles is of great importance for providing energy for
severe activity under stress conditions. At the transition of the postabsorptivc state to
the absorptive state or from muscle activity to rest, the system returns to its original
state. Adenylate cyclase and phospholipase C arc inactivated, cAMP is destroyed
by phosphodiesterase, and phosphoprotcin phosphatase causes the transition of
all intracellular enzymes of the ^cascade» to the dcphosphorylatcd form. Thus, the
regulation of synthesis and breakdown ofglycogen in the liver maintains the constancy
of the blood glucose concentration (13—5.5 mmol/l). The regulation ofglycogen
metabolism in muscles provides the energy for both intensive muscle work and energy
consumption at rest.

6,1 GLYCOGEN STORAGE DISEASES

^Glycogen storage disease* is a generic term Lo describe a group of inherited
disorders characterized by deposition of an abnormal tissue concent ration or structure
(or both) of the glycogen molecule caused by mutations in the genes that code for
enzymes involved in the synthesis or breakdown ofglycogen, as well as in the regulation
of these processes. The disorders of glycogen metabolism may alTect primarily the
liver the muscle, or both and the kidneys. The types of disorders have been described
and classified on the base of the enzyme deficiency and the affected tissue (Examples
of disorders arc shown in Table 6.2).
6.7. Glycogen Storage Diseases 299

Table 6.2. Examples of glycogen si or age diseases

Typel Von Gierke’s disease Deficiency of glucose-^ Liver cells and renal tubule
phosphatase cells loaded with glycogen.
Hypoglycemia, lactic acidemia,
ketosis, hyperlipidemia.

Type II Pompe's disease Deficiency of Fatal, accumulation of gkycogen

lysosomal a->4 and in lysosomes, heart failure.
1->6-glucosidase
(acid maltase)

Type HI Limit dextrin osis. Absence of debranching Accumulation of a characteristics

Forbes’or Con s enzyme branched polysaccharide.
disease

Type IV Amylopeciinosis, Absence of branching Accumulation of a polysaccharide

Andersen s disease enzyme having few branch points. Death
due to cardiac or liver failure in
first year of life.
Type V Myophosphorylase Absence of muscle Diminished exercise tolerance;
deficiency McArdle’s phosphorylase muscles have abnormally high
syndrome glycogen content (2.5-4,1 %).
Little or no lactate in blood after
exercise.
Type VI Hersdisease Deficiency of liver High glycogen content in liver,
phosphorylase tendency toward hypoglycemia.
Type VII Tarui's disease Deficiency of As lor type V but also possibility
phosph olructokinase in of hemolytic anemia.
muscle and erythrocytes

Type VIII Deficiency ol liver As lor type VI

phosphorylase kinase

Diseases of glycogen accumulation are caused by defect of the enzymes involved

in the breakdown of glycogen. If glycogen mobilization is impaired, then glycogen
accumulates in cells in large quantities, which can lead to cell destruction. Diseases
arc manifested by an excessive accumulation of glycogen in the liver cardiac and
skeletal muscles, kidneys, lungs, and other organs. Accumulated glycogen may be
cither normal or abnormal in. structure. The result of the disturbance of the glycogen
breakdown is hepatomegaly (liver enlargement) in some, hypoglycemia, and muscle
weakness.
Gierke's disease (type I) is noted as the most common glycogen storage disease.
A description of the main symptoms of this type of glycogenosis and their causes
may serve as a basis for understanding the symptoms of all other types. The cause of
this disease is a hereditary defect of glucose-6-phosphatase, an enzyme that ensures
the release of glucose into the bloodstream after its release from the liver glycogen.
Gierke’s disease is manifested by hypoglycemia, hypcririacylglycerolcmia (increased
level of triacylgiycerols), hyperuricemia (increased level of uric acid).
Hypoglycemia is a consequence of the violation of free glucose formation from
glucosc-6-phosphalc. In addition, due to a defect of glucose-6-phosphatase, there
300 Chapter 6. Carbohydrate metabolism

is accumulation of liver glucose-6-phosphate, which turns into pyruvate and lactate

during catabolism. The blood lactate concent radon increases and acidosis is possible.
In severe cases, hypoglycemia can lead to seizures. Hypoglycemia is accompanied bv
insulin level decrease and insulin/gtucagon ratio decrease, which leads to lipolysis
acceleration in adipose tissue as a result of glucagon action and release of fatty acids
into the blood (sec Ch. 7).
Hypcnriacytglyccrolcmia occurs as a result of a decrease in the adipose tissue
LP-lipase activity, an enzyme activated by insulin and ensuring the storage of
triacylglycerides (TAGs) by adipocytes (sec Ch. 7).
Hyperuricemia occurs as a. result of:
► increase in both glucose-6-phosphate concentration in cells and glucose-6-
phosphate conversion in the pcntose-phosphaic pathway to ribosc-5-phosphatc,
a substrate for the purine nucleotide synthesis;
* increase in the uric acid formation due to excessive synthesis, and consequently,
catabolism of purine nucleotides, the end product of which is uric acid:
► reduced uric acid excretion due eo lactate production increase and changing
urine pH value to more acidic level, that reduces excretion of poorly soluble
urates, uric acid salts.
The diagnosis is confirmed with a liver biopsy and measuring the liver glucose-6-
phosphatasc activity. In addition, a test with glucagon or epinephrine stimulation is
used. A negative lest result means that after a hormone injection the blood glucose
level changes slightly in the case of the disease.
Type I is treated with a special diet in order to maintain normal glucose levels,
prevent hypoglycemia and maximize growth and development. Therefore, diet
and lifestyle changes arc made to prevent the primary concern of the disease,
hypoglycemia. The fasting should be avoided, and frequent small meals rich in
complex carbohydrates along with fiber is recommended. It is also recommended
to limit the intake of sucrose and lactose, since galactose and fructose formed from
these di saccharides arc converted to glucose-6-phosphatc, that leads to further
accumulation, of glycogen.
Type I is inherited in an autosomal recessive manner and usually begins at three
to four months of ago. Symptoms of the disease include enlargement of the liver
(hepatomegaly), kidney (ncphromcgaly and seizures caused by repeated episodes of
hypoglycemia). Laboratory abnormalities arc elevated: levels of lactate, uric acid and
lipids (both total lipids and triglycerides). Continued low blood glucose can lead to
delayed growth and development, and muscle weakness. Affected children typically
have doll-like faces with fat checks, relatively thin extremities, short stature, and
protuberant abdomen.
The described disease is sometimes referred to as type la glycogenosis. as there
is a type lb. Glycogenosis lb is a rare pathology with the defect of glucosc-6-
phosphate translocase, which, ensures the transport of phosphorylated glucose to the
endoplasmic reticulum (ER). Therefore, despite the sufficient activity of glucosc-6-
phosphatasc, the cleavage of inorganic phosphate and the release of free glucose into
the blood is disturbed. The clinical picture of type lb glycogenosis is the same its with
glycogenosis la.
6.7. Glycogen Storage Diseases 301

Cory disease (type III) is quite common. Il is 1/4 ofal I cases of hepatic glycogenosis.
The accumulated glycogen is abnormal in structure, since the enzyme amylo-1,6-
glucosidasc (the debranching enzyme), which hydrolyzes glyoosidic bonds at branch
points, is defective. The low’ blood glucose love! manifests itself quickly because
glycogenolysis proceeds with an insignificant rate. Unlike type 1 glycogenosis, lactic
acidosis and hyperuricemia arc not observed. The patients with type 111 have a. milder
disease course.
Andersen’s disease (type IV) is an. extremely rare autosomal recessive disorder
caused by deficient activity of the glycogen-brane hi ng enzyme, amylo-1.4-1,6-
glucosykransfcrasc and results in accumulation of abnormal glycogen in the liver,
muscle, and other tissues. The structurally abnormal glycogen has few branch points,
as well as very long and sparse lateral branches that prevents glycogen to be degraded.
At the same time, hypoglycemia is moderately expressed. The disease develops
quickly, is aggravated by early cirrhosis of the liver and is practically not amenable to
treatment. Defect of the branch enzyme is found in the liver, in leukocytes, muscles,
and fibroblasts. Early and prevailing manifestations of the disease arc due to impaired
liver function.
Hers’s disease (type VI) is characterized by mild clinical manifestations and a benign
course. This glycogenosis is a consequence of the hepatic glycogen phosphorylase
deficiency. Glycogen of the normal structure accumulates in the hepatocytes. The
course of the disease is similar to glycogenesis type I. but the symptoms, hepatomegaly,
growth retardation, and mild episodes of fasti ng hypoglycemia during early childhood,
arc less pronounced. Reduced glycogen phosphorylase activity is also found in
leukocytes. Hers disease is a rare type of glycogenosis inherited in an autosomal
recessive manner.
Defect of kinase phosphorylase (type IX) is found in boys only, because the enzyme
deficiency is inherited in an X-1 inked manner.
The defect of protein kinase A (type X), as well as the defect of phosphorylase
kinase, manifests symptoms similar to Hers’s disease.
Muscle glycogenosis arc characterized by disorders of energy supply to skeletal
muscles. These diseases are manifested during physical activity and are accompanied
by pain and cramps in the muscles, weakness and fatigue.
MacArdle’s disease (type V) is an autosomal recessive myopathy caused by the
lack of muscle glycogen phosphorylase isozyme. Since the activity of this enzyme in
hepatocytes is normal. hypoglycemia is not observed (the enzyme structure in the liver
and muscles is encoded by different genes). Severe physical exertion is poorly tolerated
and may be accompanied by convulsions. However, lactate hyperproduction is not
observed during exercise. That emphasizes the importance of different energy sources
for muscle contraction, such as fatty acids (see Ch. 7). Although the disease is not sex-
linked, the high incidence of the disease is characteristic of men.
Phosphofructokinase deficiency is characteristic of type VII glycogenosis.
Patients can. perform moderate exercise. The course of the disease is similar to type V
glycogenosis, but the main manifestations arc less pronounced.
The defect of phosphoglyccrate mutase and the defect of the M-subunit of LDH
are characteristic of muscle glycogenosis. Manifestations of these pathologies are
302 Chapter 6. Carbohydrate metabolism

similar to Mac Ardle’s disease. The defect of phosphoglycerate mutase in the muscles
is described in one patient only.
Diseases are associated with disturbance of glycogen synthesis (e.g., deficiency of
glycogen synthase) and are accompanied by a decrease in its content in the tissues. The
most characteristic symptom of such diseases is acute hypoglycemia (since there are no
glycogen stores), especially after a night fasting. Hypoglycemia can lead to vomiting,
convulsions and loss of consciousness. Constant starvation of the brain leads to a delay
in menial development. The majority of these patients die in early childhood: frequent
feedings can significantly reduce the symptoms of the disease.

6.8. THE METABOLISM OF ETHANOL IN THE LIVER

The metabolism of ethanol includes dehydrogenation reactions, which, result in
the formation of acetic acid (Fig. 6.30). Ethanol is a small two carbon alcohol that,
due to its small size and alcoholic hydroxyl group is both lipid- and water-soluble. Il
is readily absorbed! from the inLestinc by passive diffusion, enters the blood, and is
mainly metabolized in the liver.

CH3CH£OH
E tnan:

O
Ch:C
H
Afraid my de;

NAD*

NADH + H +

; Acetate I

fip. 6.30. The pathway of ethanol metabolism in the liver. ADM, alcohol be hydrogenase: ALDH,
acetaldehyde dehydrogenase

The most significant pathway of ethanol metabolism in the liver is through the key
enzyme in alcohol metabolism, liver alcohol dehydrogenase (ADH). The ADH is a
cytosolic NAD4-requiring enzyme expressed at high concentrations in hepatocytes.
It oxidizes ethanol io acetaldehyde with, reduction, of NAD* to NADH (Fig. 6.30).
Approximately 90% of the acetaldehyde that is generated is further metabolized
to acetate in the liver. The major enzyme involved is a low Km mitochondrial
6.3. The Metabolism of Etnanoi tn the Liver 303

acetaldehyde dehydrogenase (ALDH), which oxidizes acetaldehyde to acetate with

generation of NADU. Acetate may be activated to acetyl coenzyme in the liver but
most of the acetate that is generated enters the blood and is activated to acetyl-CoA in
skeletal muscles and other tissues where it can enter either the tricarboxylic acid cycle
and is eventually metabolized to carbon, dioxide and water or the pathway for fatty acid
synthesis. This is the main metabolic route of ethanol in the body; partially; ethanol
is oxidized in other ways independent of AD H via the microsomal ethanol oxidizing
system.
As a result of the rapid dehydrogenation of large amounts of ethanol in. liver, the
NAD concentration decreases and NADU concentration increases. The oxidation
of the daily requirement of carbohydrates (300-400 g) requires the same amount of
NAD’ as the oxidation of - 125 g ofethanol. After a meal, much of the dietary glucose is
deposited in the form of glycogen and is consumed gradually. The ethanol metabolism
occurs in a significantly shorter time. Therefore, in the case of ethanol consumption
exceeding the definition, of moderate alcohol drinking, the ratio [NAD ]/|NADH|
decreases. The rate of the reactions that arc dependent on NAD and NADH changes.
In particular, stationary concentrations of pyruvate and lactate change:

Pyruvate + NADH + H' -*---------- ► Lactate + NAD

The pyruvate concentration in the cells and in the blood decreases, and the lactate
concentration, increases. As pyruvate is a precursor of the gluconeogenesis, the rate
of the pathway in the liver decreases and hypoglycemia develops. Especially acute
hypoglycemia occurs under lack of glycogen stores in the liver and muscles (after
taking alcohol on an empty stomach, after considerable muscle activity, in chronic
alcoholics, who have constantly decreased appetite). Hypoglycemia can cause a loss
of consciousness due to alcohol poisoning. With high doses and chronic alcohol
consumption, a lot of blood acetaldehyde damages cell membrane structures. In
particular, mitochondria arc damaged: the transmembrane potential and the efficiency
of coupling of respiration and phosphorylation arc reduced.

The quantitative characteristics ol the metabolism of carbohydrates studied in this unit

blood glucose concentration: 3 3-5.5 mmol/L 80-100 mg/dL

Review tests
I. Mulch the figure and the Idler.
The scheme of glycogen degradation:
Enzyme:
A. Phosphoglucomuiasc.
B. a1.6-glycosidasc.
C. Glycogen phosphorylase.
D. Phosphatase.
E. Ofigosaccharyl transfcrase.
304 Chapter 6. Carbohydrate metabolism

esecooccooc

*
■
■

I
Gl ucoso-6-phosphate
2. Match the figure and the letter.
The diagram of glycogen degradation:
Metabolite:
A. Glucose.
B. ATP
C H/O^
D. ADP.
E. Glucose I-phosphate.

eiexcaxcc

*
I

I
Glucose-6-phosphate
6.3. The Metabolism of Ethanol tn the Liver 305

J. Match the figure and the letter.

Regulation of glycogen synthase and glycogen phosphorylase activities:
Enzyme:
A. Active glycogen synthase.
B. Inactive glycogen phosphorylase.
C. Phosphoprolcin phosphatase.
D. Active glycogen phosphorylase.
E. Inactive glycogen synthase.

ATP -h.
2 Pi Pi
— Glycogen phosphorylase-OH h/l\rC □lycogen synthase-OH 1
^-ATP
I
___________________________________ I
ADP-' * Glycogen phosphoiylase®^ Glycogen synthase® * * ADP

4. Match the figure and the letter.

Regulation of glycogen synthase and glycogen phosphorylase activities:
Enzyme:
A. Active glycogen phosphorylase.
B. Phosphorylase kinase.
C. Protein kinase A.
D. Phospho prole in phosphatase.
E. Inactive glycogen synthase.

Pi Pi
Glycogen phosphorylase-OH p / X 4 Glycogen synthase-OH “

3- ,
ATP ATP

ADP Glycogen phosphorylase® *- . Glycogen synthase® ADP

1 JL 3

Situational Problems
I. During an exam a student’s blood glucose was 7 mmol/l. Explain the reason Tor
the observed change in blood glucose if the student had breakfast 4 hours before
the exam. For answer:
a) indicate the normal blood glucose concentration:
b} name the hormone which concentration rises in the student’s blood in this
situation:
c) draw a chart of the process activated in the liver by this hormone and indicate
the regulatory enzyme:
d) draw the scheme ofhormonal signal transduction and describe the mechanism
of the hormone effect on the regulatory enzyme.
306 Chapter 6. Carbohydrate metabolism

2. A person performs intense exercise (for example, runs away from danger)
30 minutes after a carbohydrate-rich lunch. Why docs the glycogen synthesis
stop in skeletal muscles in th is situation? Why is glycogen breakdown stimulated?
To answer the questions:
a) write a diagram of glycogen synthesis, specify die reactions associated with
energy consumption when a glucose molecule is included in a growing
glycogen chain:
b) write the glycogen mobilization diagram and calculate the ATP amount that
can be produced if glucose I-phosphate is further oxidized to CO3 and H J)
in the muscles;
cl indicate the hormone with an increased level in blood under stress situations,
and how this hormone affects the activity of regulatory enzymes of glycogen
synthesis and breakdown.
3. Alkaloid caffeine contained in coffee seeds causes hyperglycemia and has a
stimulatory effect, although it does not interact with epinephrine receptors.
It is known that caffeine inhibits phosphodiesterase activity. Why can caffeine
decrease the blood glucose level? Explain the answer and draw the appropriate
schemes.
4. A patient had severe fasting hypoglycemia. The liver biopsy showed that
glycogen synthesis occurs, but that liver tissue accumulates molecules with short
side branches. What enzyme deficiency can cause this pathology? Explain your
answer by writing a diagram of the process that should occur normally. Indicate
the site of the disturbance on the diagram.
5. Popular ^energy* drinks arc nonalcoholic carbonated beverages. Advertising
emphasizes that they increase working capacity. Most beverages contain, caffeine
and its analogs, theobromine and theophylline (vegetable alkaloids), vitamins,
easily digestible sugar (glucose, sucrose), taurine, carnitine. These drinks arc
highly carbonated, which accelerates their absorption into the blood. Why do
energy drinks improve efficiency? For answer:
a) draw a diagram of the glycogen metabolism pathway with its the acceleration
possibly contributing to belter efficiency:
b) indicate the mode of caffeine action on the rate of this process, if it is known
that it inhibits phosphodiesterase;
c) explain why 3-4 hours after the taking of ^energetics* fatigue and drowsiness
occur.
6. In an experiment, students used liver tissue samples to study the ethanol
metabolism and the possibility of ethanol conversion into glucose. The ethanol
introduction in the investigated medium didn’t lead to glucose level increase.
Why is it impossible to convert ethanol to glucose? For the answer:
a) provide a scheme of gluconeogenesis, indicate the substrates of this process;
b) write the reaction of ethanol oxidation in the liver:
c) explain whether it is possible to use the metabolites of ethanol catabolism for
the glucose synthesis.
7. In experimental laboratory, the effect of epinephrine on glycogen metabolism
in a muscle tissue sample of the mu Lam mice line was studied. After adding
6.3. The Metabolism of Ethanol tn the Liver 307

epinephrine to the investigated medium cAMP concentration in the cells

increased, but glycogen phosphorylase was present only in. de phosphorylated,
inactive form, (jive the enzyme defect most likely in the mutant mice muscles?
To solve the problem, answer the questions:
a) can these animals perform sudden, intense work, for example, to run away
from danger?
b) can these animals do moderate physical activity?
c) argue the answers by making a scheme of the hormone action on glycogen
metabolism in the muscles.
S. Hers disease (type IV) is a rather rare type of glycogenosis inherited in an
autosomal recessive manner. In early childhood, patients with Hers disease
VI usually present with hepatomegaly mainly caused by a defect in. glycogen
phosphorylase. Explain why a glycogen phosphorylase defect leads to an increase
in liver and hypoglycemia? For the answer:
a) name the hormones stimulating glycogen breakdown and indicate different
mechanisms of signal transduction by these hormones;
b) write a diagram of the process that is disturbed in patients, indicate the
enzymes involved in this process, and answer the main question of the
problem:
c) explain the causes of glycogenosis conditions, give their classification.
9. In a patient with a hereditary disease caused by liver phosphorylase kinase defect,
hepatomegaly is observed (an increase in liver size), glycogen of the normal
structure accumulates in the liver cells, moderate hypoglycemia is observed
3 hours after a meal. Explain the listed symptoms and the accumulation of
unchanged structure glycogen in this pathology. For this:
a) draw a process diagram in which phosphorylase kinase is involved:
b) indicate the metabolic block on the diagram and explain the symptoms
observed in the patient.
10. Glycogen, as the main storage form of glucose, is an important energy reservoir.
Describe the role of glycogen, in providing the body with energy. To answer:
a) write a scheme for glycogen mobilization. and oxidation of the end product
to CO2and H?O;
b) mark the reactions associated with ATP synthesis and ATP consumption in
the scheme, calculate the oxidation energy yield of I mole of the final product
resulting from glycogen breakdown;
11. An unconscious man with signs of alcohol poisoning was taken to the hospital.
The laboratory data of blood tests are the following:
a) alcohol — 320 mg / dl (the norm — 5 mg/dl);
b) glucose — 50 mg/dL;
c) lactate — 2 mmol /1 (the norm — I mmol/l).
What arc the causes of changes in blood glucose and lactate concentration in
acute alcohol poisoning? Draw the appropriate reaction and the diagram of the
process with a reduced rate in this person.
 Chapter 7
LIPID METABOLISM

7.1 Structure and Functions of the Major Human Lipids

72. Digestion and Absorption of Dietary Lipids. Transport of Lipids by Chylomicrons
73. Disorders of Lipids Digestion and Transport
7.4. Synthesis of Fatty Acids and Triacylglycerols - Lipogenesis, Hormonal. Regulation.
Obesity
7.5. Mobilization of Stored Fats from Adipose Tissue
7.6. Utilization of Fatty Acids for Energy Production - p-oxidation. Disorders
of fJ-oxidation. The Other Pathways of Fatty Acids Oxidation
7.7. Synthesis and Oxidation of Ketone Bodies. Ketonemia, Ketoacidosis
73. Structure Metabolism and Biological Effects of Eicosanoids
7.9. Glycero phospholip ids and Sphingolipids, Biological Functions and Metabolism
7.10. Cholesterol Functions and Metabolism. Regulation of Cholesterol Synthesis
7.11. Bile Salts, Functions, Synthesis, and Role in the Cholesterol Metabolism
7.12. Cholesterol Transport by Blood Lipoproteins
7.13. Hypercholesterolemia. Biochemical Aspects of Atherosclerosis.
Ch oleste ro I- Lowe ri ng Agents

7.1 STRUCTURE AND FUNCTIONS OF THE MAJOR HUMAN LIPIDS

Lipids arc organic molecules with very diverse chemical structures and biological
functions, but they have one physical property in common: relative insolubility
in waler. The latter governs their unique functions. Lipids arc the major structural
compounds of all membranes in all living systems, serve as fuels and hormones and
hormone-Like signal molecules.
Lipids include the following classes: fatly acids, triacylgiycerols, phospholipids,
sphingolipids, glycolipids, steroids, fat-soluble vitamins, eicosanoids (Table 7.1).
Fatty acid consists of an alkyl chain with a terminal carboxyl group (Fig. 7.1). Most
of the lipids found in the body contain, fatty acids bound by an ester bond to hydroxyl
group of glycerol or cholesterol. Most of the laity acids in the human lipids have an
even number of carbon atoms, usually between 16 and 20 (Table 7.2). The carbon
atoms of the fatly acid arc numbered starting with the carboxyl carbon. Alternatively,
carbon atoms arc designated by Greek letters. The o-carbon atom is the next to the
carboxyl carbon, fi-carbon atom is the next to ci-carbon atom and etc. The last carbon
atom in the chain is the co-carbon. The designations are needed for the classification
of fatty acids. Fatty acids may be saturated and unsaturated. Saturated fatty acids
have no double bonds in the chain. Their general formula is CH -(CH j -COOH.
7.1. Structure ana Functions of me Major Human Lipids 309

Table 7.1. Structures, functions ana location of lipins in humans

CLASS OF UPlDS STRUCTURE FUNCTIONS LOCATION

All cells
Structural compounds
(as me compounds
Fatty acids R-COOH of lipids, fuel of other lipids — TaGs.
molecules
phospholipids)

0
Long — term storage
HbC-O-4-R.,
Triacyglycerots of energy, tnermo-
and mechanic Adipose cells
(TAG) R^-C-O-CH 0
protection
HjC -- 0—C "Rg. of a body
6 Structural components
0 H^C-O-A-R, of membranes,
Glycerophospholipids
X: chorine, monolayer Cell membranes,
ethanolamine, fu-A-o—Ah o on the surface lipoproteins, alveoli
inositol bisphosphale. of lipoproteins, of me lungs, bile
HjC-D-P-O-X
serine amphipathic compounds
Ah of bite micelles

| Ceram tdej

O A component of
Spliingophospholipkis the myelin sheath Central nervous system
(Sphingomyelin) O-P-O around neurons
1.
Chains

Sphingolipids:
I Ceramide ] intercellular Membranes of Che cells
Cerebrosides —
1 communication, and the myelin sheath
X-one monosaccharide.
Carbohydrate-X antigenic of the central
Gangl iosides-X-complex
determinants nervous system
sugars
The component
of me ceHs membranes
Cell membranes,
Steroids Cholesterol the precursor of bile lipoproteins, bite
salts and steroid
hormones

Regulators of the
Derivatives of 20 carbon Almost every cell
Eicosanoids cellular functions,
polyunsaturated fatty acids of the body
‘Local’ hormones

Palmitic (16:0) and stearic (18:0) acids arc the most common saturated fatty acids
in humans. The numbers 16 or IS denote the number of carbon atoms in the fatty
acids. Zero denotes that these fatly acids don't contain double bonds. Saturated fatty
acids account, for up to 40-45% of the fatty acids in human fat in adipose tissue.
Unsaturated fatty acids contain one or more double bonds. The most commonly used
systems designating the position of the double bonds in unsaturated fatty acid arc the
della (A) numbering system and cn-system (Table 7.2, Pig. 7.1). For example, 18:1A9
denotes that the fatty acid contains one double bond between the ninth and tenth
carbon atoms starting from the first carbon of the carboxyl group. The same fatly acid
is designated as 18:Iu>9 that denotes the distance of the double bond closest to the
<o-cnd (the lasL methyl group in an aliphatic chain of fatly acid).
310 Chapter 7. Lipid metaD ol ism

Carboxyl
group

Hydrocarbon
chain

(a) (b)

Mixiure of saturated and

unsaturated Fatly acids

W)

Fig. 7.1 Structure ol saturated and unsaturated fatty acids: a) saturated fatly acid; b) monoenic fatty
acid wim one double bond in me cis conformation; c) orderly packaged saturated fatty acids in lipid
phase, solid at room temperature; di fatty acids with a lower melting point are fluid al body temperature

The double bonds arc usually in the cis conformation of the native fatly acids. Cis
conformation induces the kink in the fatty acid chain, preventing orderly packing
of the chains in lipid phases ( Fig. 7.1). The melting point of a fatty acid decreases
with the degree of saturation and increases with chain length. As a result, the double
bonds increase the fluidity of lipids, containing unsaturated fatty acids. Thus, the fatly
acids composition of membrane phospholipids determines the fluidity of the inner
hydrophobic layer of membranes at body temperature.
7.1. Structure ana Functions of me Major Human Lipids 311

Table 72. Tne main fatly acids of the humans. Designation of fatty acids by delta (A) and n/-systems:
16 denotes the number of carbon atoms in the fatty acid: 0 - fatty acid doesn't contain double bonds
(16:0 palmitic acid): 20:4A5,8,11J4-arachiconic acid contains 20 carbons and four double bonds at the
5* S* 11*14" carbon atoms starting from me 1st carbon of carboxyl group: ’ - essential fatty acid

Palmitic 16:0

Stearic 16:0

Palmitoleic 16:1 A9 9

Oleic 18:1 A 9 9

Linoleic 18:2 A 9.12* 6

■3
a-Linolenic 18:3 A 9,12,15*

Eecosatrienoic 20:3 A 8,11,14 6

Arachidonic 20:4 A 5. 8,11,14 6

Eecosapentaenoic 20:5 A 5. 8,11.14,17 3

Fatly acids arc subdivided into nonessential and essential by their requirement to
humans. Noncssenlial laity acids arc saturated or nionocnic fatty acids (contain one
double bond) and synthesized in humans. These acids don’t have to be obligatorily
included in the diet. Polyunsaturated fatty acids arc essential and belong to two
series. One is w6 like linoleic (18:2 ci>6) and arachidonic (20:4co6) acids. Another is
w3 like a-linolcnic (I8:3 cd 3) acid (Fig. 7.2). Linoleic and n-linolcnic acids arc not
synthesized in mammalians as they lack enzymes necessary to insert double bonds
beyond the 9th carbon and w-carbon of fatty acids. These fatty acids must be in the
diet and can be obtained from plant oils or cold-water fish oil. The fish obtain these
fatly acids by consuming water plants. Linoleic acid can be converted to arachidonic
acid, whereas a-linolcnic — to cicosapentaenoic acid (20:5 A5,S,I I.14J7) in the body.
These 20 carbon fatty acids are the precursors of prostaglandins and other eicosanoids
which arc important in cell communication. Another important function of linoleic
acid is Lo form unusual lipid in the skin that helps to make the skin impermeable to
water. Dietary deficiency of essential fatty acids results in dermatitis.
Tri acylglycerols, more commonly known as fat, are the major lipids in human
body. Triacylglyccrol contains glycerol in which all three hydroxyl groups are cstcrificd
with fatty acids (Table 7.1). Most native triacylglyccrols have different fatty acids
cstcrificd to the three positions of the glycerol, the most unsaturated fatty acid is
usually bound with 2nd carbon of glycerol. The distribution of the fatty acids across
those three positions of the glycerol moiety of triacylglyccrol is influenced by many
factors., including diet and anatomical location of the stored molecule.
Triacylglyccrols arc stored in adipocytes of adipose tissue specialized lor long —
term energy storage. Adipose fat is very efficient fuel storage: triacylglyccrols contain
more calorics per gram than carbohydrate or protein (9,3kcal/g for fats as opposed to
4,0 kcal/g for glycogen), because they are the most reduced organic molecules in a
body and triacylglyccrols drop in adipocytes do not contain water. A normal man of
70 kg weight has about 15 kg stored triacylglyccrols in adipose tissue, which accounts
312 Chapter 7. Lipid metaD ol ism

for - 85% of his total stored calorics. Subcutaneous adipose tissue is also important in
temperature regulation because it provides a layer of insolation.

AAAA/VW”" Linolsic acid (m6.1&24*12)

18
/uVuVVV\ 15 12 9
a-Linolsic acid (m3. 10:3^12,15)
COOH

20 17 14 11 3 5 COOH
Elcosapentaonoic acid (m3. 20:5A5,8,11,14,17 )

Fig. 72. The structures of the polyunsaturated fatty acids. denotes the position of me closest
double bond to m end (methyl end) of fatty acid; me same for qj3 and gj9 fatty acids: w6 linoleic (182)
and iu3 a-iinoienic n 8:3) acids are essential for humans

Glycerophospholipids (phosphoacylglycerols) have glycerol as a backbone, two

fatty acids cste rifted to position I and 2 of glycerol and a phosphate (alone or with,
substituent) attached to the 3d carbon, of glycerol. These lipids arc amphipathic
molecules because they contain both polar and nonpolar regions. The main
glycerophospholipids arc phosphatidylcholine (lecithin), phosphalidylei ha nolam inc,
phosphatidyl inositol (Table 7.1). Glycerophospholipids arc the structural components
of all types of biological membranes and phospholipid monolayer on the surface of
blood lipoproteins. Dipalmiloyl phosphatidylcholine has a special function as the
major compound of pul mo nary surfactant. The surfactant is secreted by alveolar cells
and reduces the surface tension of the film that lines the alveolar walls. Without it, the
alveoli collapse and breathing become difficult.
Sphingolipids — arc lipids, which have amino alcohol sphingosine backbone.
If sphingosine would bind a long-chain fatty acid through its amino group, then
ceramide can be formed. Sphingomyelin is the phospholipid because it contains the
phosphocholinc attached to the hydroxyl group of ceramide. Other sphingolipids
7.2. Digestion and Absorption of Dietary Lipids. Transport of Lipids try Chylomicrons 313

include glycolipids (gangliosides, galactoccrcbroside, glucoccrebroside) containing

one or more molecules of sugars: glucose, galactose.
Sphingomyelin is the main compound of myelin sheath around neurons;
gangliosides and galactoccrcbroside are important for formation of the brain grey
matter. Glycolipids comprise antigenic determinants of blood groups (ABO).
Cholesterol contains four rings and aliphaLic side chain. It is the rigid structural
compound of biological membranes and the precursor for the synthesis of bile salts
and steroid hormones.
Eicosanoids are derived from polyunsaturated fatty acids containing twenty carbon
atoms like arachidonic acid (Fig. 7.2). The eicosanoids arc a large group of hormone-
like signal molecules produced by many cells in the body. They arc involved in the
regulation of many physiologic reactions in humans.
Fat-soluble vitamins arc essential compounds and include vitamins: A — required
for vision, I) — the precursor for the synthesis of the hormone regulating calcium
metabolism, E — antioxidant, K — participant of blood coagulation system.

72. DIGESTION AND ABSORPTION OF DIETARY LIPIDS. TRANSPORT

OF LIPIDS BY CHYLOMICRONS
Lipid requirements for adults arc about 80— tOO g per day. About 90% of
dietary lipids arc triacylglycerols (TAG), - 10% arc cholesterolT cholesterol esters,
phospholipids. Half amount of dietary fats should be oils containing essential fatty
acids which arc not synthesized in mammalians.
Digestion of dietary lipids in adultsstartsinthc small intestine (Fig. 7.3)..Al first, very
acidic content which enters from stomach into the small intestine, is being neutralized
by bicarbonate of pancreatic juice. As a result, the pH of this mass rises up to neutral
meanings, and this is optimal pH for the action of all digestive enzymes of the intestine:

H +HCO1 -H3CO-H2O+CO3r

The next step is the emulsification of lipids. The step is required for the conversion
of big waler-insoluble fat particles to microscopic particles, so fat would become more
available for the action of digestive enzymes. Fat emulsification is carried out by bile
salts, which arc synthesized from cholesterol in the liver and arc secreted into the small
intestine from gallbladder where they are stored. The contraction of the gallbladder
and secretion of pancreatic lipase arc stimulated by the gut hormone cholecystokinin.
Bile salts, as amphipathic compounds, possess both, hydrophobic and hydrophilic
pans and act as detergents. They surround the lipid drop so that hydrophobic pans arc
immersed into lipid drop while hydrophilic parts arc on the surface of the drop. A big
lipid drop then divided into small droplets, as the surface tension lowers. Peristalsis
of duodenum and the presence of CO. arc also involved in small droplets formation.
Emulsification enormously increases the availability of lipid molecules for the action of
water-soluble enzyme — pancreatic lipase, because lipase acts on the oil-water interface.
The fat digestion is catalyzed by pancreatic lipase, which is secreted from pancreas.
The small protein — colipase — is needed for the activity of pancreatic lipase. Colipase
is secreted also from pancreas and is activated by trypsin through, partial cleavage.
Colipase stabilizes and activates pancreatic lipase improving the interaction of the
314 Chapter 7. Lipid metaD ol ism

enzyme with water-insoluble cm nisi tied fat drops. The pancreatic lipase hydrolyzes
triacylglyccrols by removing fatty acids from the 1st and 3d carbons of glycerol and
producing two fatty acids and 2-monoacylglyccroL

Dietary
TAG (lipidsj

Bile salts Gallbladder

Digestion of fats
^mulsifjcation. hydrolysis) ^HCQa-

Emulsified TAG
oooo Pancreas
oooo
Pancreatic lipase

Colipase ]-*
o
£
E
3
fl)
JZ

Mixed micelle

Resynttiesis of TAG

2 acyl-CoA
h2COH H3C O- CO Rf

u p?—co— o— Ch — Rj— CO— O—C H

fl)
J=
Hgp—OH F^c-O— CO— Rs
-I— 2 CoASH
□l

Formation of nascent
chylomicrons

Formation of mature
chylomicrons

Q
m

Fig. 7.3. Digestion and absorption ol dietary triacylglycerols. Packing of lipids into chylomicrons
7.2. Digestion ana ADsorplioa of Dietary Lipids. Transport of Lipids Dy Chylomicrons 315

Another two lipases exist in humans — lingual and gastric, but they digest mainly
emulsified fat of milk and arc important for infants. In adults these enzymes digest
not more than 15% of dietary triacylglycerols. Cholesterol esters present in dietary
lipids arc hydrolyzed by pancreatic cholesterol ester hydrolase (cholesterol esterase)
that produces cholesterol and fatty acid. The degradation ofglyccrophospholipids is
performed by phospholipases of pancreatic juice. Phospholipase A, is activated in the
intestine by trypsin and removes the fatty acid from position 2 of the glycerol, leaving
a Ivsophospholipid as a result.
The products of triacyl glycerols digestion — fatty acidsand 2-monoacylglyccrols —
arc packaged into mixed micelles, that help hydrophobic molecules pass through the
membranes of intestinal epithelial cells. Other dietary lipids — like cholesterol and
fat-soluble vitamins arc also packaged in the micelles stabilized by the bile salts. The
micelles arc water-soluble because the hydrophilic groups of bile salts, fatty acids,
2-monoacylglyccrols and cholesterol arc exposed to water a nd arc outside Lhe micelles,
but the hydrophobic are inside. The micelles travel to the microvilli of the intestinal
epithelial cells, where the products of lipids digestion arc absorbed but the bile salts
arc left behind in the In men of the gut and arc intensively resorbed, when reaching the
ileum (recycling of bile salts, Ch. 7.4).
The absorbed fatly acid is activated to acyl-CoA by acyl-CoA synthetase in
endoplasmic reticulum of the intestinal mucosal cells:

ATP

FACoA.

This is the first reaction of a fatty acid after entering a cell, like the reaction of
glucose phosphorylation by hexokinasc or glucokinase is always the first reaction of
intracellular metabolism of glucose. Like glucose 6-P, acvl-Co A cannot cross the
plasma membrane and is used only in intracellular metabolic pathways. The acyl-
CoA derived ITom the absorbed fatty acid reacts with 2-monoacylglycerol to form
at first diacylglycerol (DAG), then another fatly acyl-CoA binds to DAG to form
triacylglyccroL This process is called the resynthesis of triacyEglycerols:

Apc^raLein B43

Nascent
chyJonycrons

CoASH
'2 Monoacylgly-zerol] Triacylglycerol

High, insolubility in. water of the synthesized TAGs and other absorbed lipids
leads to their coalescence in blood, impeding the blood flow: this explains why the
lipids are transported by blood in water-soluble lipoproteins particles. Intestinal
cells package triacylgiyccrols and other hydrophobic molecules in lipoproLcins —
chylomicrons (CM). Lipoproteins arc composed of neutral lipid core surrounded
by a shell of compounds oriented so that their polar portion is exposed on the
316 Chapter 7. Lipid metaD ol ism

surface of the lipoproteins (Fig. 7.4). The protein compounds of lipoproteins arc
named apoproteins. They arc different in different types of lipoproteins (Table 7.3).
The major protein compound of chylomicrons is apoprotein B-48. This apoprotein
is coded by the same gone as apoprotein B — 100, which is synthesized in the liver
and serves as the major pro Lein compound ofVLDL. In the intestine, a stop codon is
generated during posttranscriptional modification ofmRNAand the protein which
is synthesized comprises only 48% of the size of the protein produced in the liver;
hence the designations are B-48 for apoprotein of chylomicron and B — I00 for
apoprotein ofVLDL. Besides apo B-48 apoprotein apo A-1 is synthesized in intestine
and included in the nascent chylomicrons. Triacylglycerols, which arc resynthesized
in the endoplasmic reticulum of intestinal cells, complex with the proteins to form
the chylomicrons. Triacylglyccrols are the major lipid compound of chylomicrons
(85%) and the function of chylomicrons is to deliver the dietary’ lipids to peripheral
tissues.
The absorbed lysophospholipids and cholesterol are converted into phospholipids
and cholesterol esters in rcacylation reactions and then, also, arc packaged in
chylomicrons. Short and medium-chain length fatty acids of milky fat arc not
convened to their CoA derivatives, but rather released into the portal circulation where
they are carried by serum albumin to the liver.

Rql TA General scheme of me structure of a blood lipoprotein. Pm - phospholipids, hydropnilic

parts are on tne surface of the lipoprotein, tiydrophobic aliphatic radicals of fatty acids are turned
into hydrophobic core of a lipoprotein. The core of lipoprotein is nydrophobic and contains TAG and
cholesterol esters. The surface of lipoprotein is covered with monolayer of pnosphetipid molecules and
hydrophilic moiety of apoproteins
7.2. Digestion and Absorption of Dietary Lipids. Transport of Lipids try Chylomicrons 317

ladle 7.3 The composition of lipoprole ins

CfrTtomicrons VLDL DL LDL fOL

Composition^^:

Proteins 2 10 11 22 50

Phospholipids 3 18 23 21 27

Cholesterol 2 7 8 8 5

Cholesterol 3 10 30 42 16
esiers

TAG 85 55 26 7 3

Functions Miter dietary Deliver lipids Intermediate Deliver Reverse

Lipids from Endogenous Density Cholesterol Cholesterol
intestine Lipids from Lipoproteins. to the transport
the Eiver Precursor of tissues From the tissues
LDL to the liver, donor
oi apoproteins

Primary tissue Intestinal The liver Blood Blood Hepatocytes:

Source epithelial HDL — precursors
Cells

Density, g/ml 0.92-0,93 0.96-1,00 1,00-1,06 1,06-1.21

Diameter, nrn >120 30-100 21-100 7-15

The main B-48 B-100 B-100 B-100 £. A-l, A-ll

Apoproteins Git, A-l Cll, E Cll. E Cll

E, A-ll E. A-l!

TAG — triacyigJyc&«M&; VlDL — vary low-dMisily lipopratflins; LOL — low-density lipoprelBins:; IDL — Intermediate density
lipoproteins; HOL — high-tfeasity lipopwtains; B — -4S structural apoprotein of chylomicrons: B —100 — structural
apoprotein of VLDL IDL; LDL; Cai — activator of lipoprotein lipase; E — mediates tha uptake nt chylomicron remnants and
iDl mainly try receprws on ins surfacB of hapafocytBs.

Nascent chylomicrons arc secreted into lymphatic system by the process of

exocytosis and then enter the blood through the thoracic duct. Nascent chylomicrons
begin to enter the blood within I hour after the start of a fatty meal. Initially, the
particles arc called nascent (newborn) chylomicrons. The nascent chylomicrons obtain
apoproteins apoCi I and apoE from HDLs within the blood and become « mature*
chylomicron. HDLs arc formed in the liver, and one of their functions is to transfer
apoproteins to the nascent chylomicrons and VLDL.
The function of chylomicrons is to deliver dietary lipids, mai nly triacylglyccrols,
from intestine to peripheral tissues — adipose tissue, heart, skeletal muscles, lactating
mammary gland (Fig. 7.5). These tissues possess the enzyme lipoprotein lipase
(LPL) that is attached to the surface of the capillary endothelium by heparan sulfate
proteoglycans. When the chylomicrons pass through the capillaries, lipoprotein lipase
recognizes apoCi I on the surface of the panicle, binds it, and active site of the enzyme
hydrolyzes the iriacylgiyccrols ofchylomicron to glycerol and 3 fatty acids. The glycerol
is transported to the liver and may be used for triacylglyccrols synthesis in fed state. The
fatty acids released from triacylglycerols of chylomicrons arc stored as fat in adipose
tissue and oxidized in the muscles generating energy. The isoenzyme of lipoprotein
318 Chapter 7. Lipid metaD ol ism

lipase synthesized in adipose tissue has a higher Km, than the isoenzyme synthesized
in muscle cells, therefore, the adipose lipoprotein lipase is more active after a meal,
when chylomicrons level increases in blood. Insulin, which secretion raises in the fed
stale, stimulates the synthesis and secretion of adipose lipoprotein lipase. This ensures
that dietary fat is directed mainly to adipose tissue in well-fed state. Lipoprotein lipase
in capillaries of the muscle cells hasa lower Km than in adipose tissue, thus, the muscle
cells can obtain fatty acids as fuel molecules from blood lipoproteins when they arc
needed for energy, even if the concentration of lipoproteins is low (fasting slate). After
realizing the triacylglycerols from chylomicrons, apoCi I returns back to HDL and
chylomicrons becomes chylomicron remnants, containing apoE. ApoE is recognized
by membrane receptors, particularly those on the surface of hepatocytes, allowing
apoE-bearing lipoproteins to enter these cells by cndocytosis for subsequent digestion
by lysosomes. Lysosomal enzymes degrade the chylomicron remnants and released
products of digestion can be reutilized by the hepatocytes (cholesterol, fat-soluble
vitamins, phospholipids, amino acids). In the capillaries of the liver, another lipase
is located - hepatic triacylglycerol lipase (HTGL). U LG L also digests triacyl glycerols
of lipoproteins, hut its substrates arc panicles already partially digested by lipoprotein
lipase. Hepatic iriacylglyccrol lipase facilitates the conversion of IDE into LDL.

Lymph
chylomicrons blood

Capillary walls
HDL
Chylomicrons
Chylomicrons
intestiaJ
epitheHal cell

Endocytic
Chylomicron
vesicle
remnants

Glycerol
Cholesterol
Receptors
Amino acids
Glicerol

of chylomicrons in transport of dietary lipids. FA—fatty acids, TG — Oiacylgtycerol,

Rg 7.5. Function
LPL— lipoprotein lipase. Cl I — apoprotein Oil, HDL — high-density lipoproteins
7 J. OfSGraers of Lipids Digestion and Transport 319

73. DISORDERS OF LIPIDS DIGESTION

AND TRANSPORT
As bile saltsand pancreatic lipase arc needed for normal triacylglyccrols digestion,
low secretion of these compounds leads to lai malabsorption, so nondigested lipids arc
excreted in feces. In case of pancreatic failure, as a. result of inflammation or due to
other diseases, low secretion of pancreatic juice, containing pancreatic lipase, colipasc,
and bicarbonate is taken place, it causes bulky, fatty, floating stools that contain
undigested triacylglyccrols. This condition is called steatorrhea. As a consequence,
some deficiencies of fat-soluble vitamins and essential fatly acids (linoleic and
linolenic) can occur for the vitamins arc excreted in feces along with the fat rather
than being absorbed. A lack of bile salts in patients with biliary obstruction has similar
symptoms, but in this case most unabsorbed compounds in the stool are fatty acids,
di- and triacylglyccrols. Pat malabsorption is treated effectively, because pancreatic
enzymesand bile salts arc available as medicines and used in patients with pancreatic
insufficiency or biliary disease.
Impaired transport of lipoproteins by blood leads to another type of disease.
Deficiency of lipoprotein lipase or its activator apoCi 1 (familial lipoprotein lipase
deficiency, dislipoproteinemia type I.) is a rare autosomal recessive genetic disorder
leading to hype rchy lorn icronemia and hypertriacylglyccrolcmia. The disease present
at birth, in which the body is unable to clear chylomicrons from the blood. Blood
tests show extremely high levels of TAG and chylomicrons. Patients have enlarged
liver and spleen and develop eruptive cutaneous xanthomas. This disorder doesn’t
lead to atherosclerosis but can cause pancreatitis which can be fatal. People who
have the disorder must avoid eating fats of all types — saturated, unsaturated, and
polyunsaturated.
Formation of chylomicrons in the endoplasmic reticulum of the cntcrocytes
and VLDLs in the liver requires the specific protein — microsomal triglyceride
transfer protein (MTP). The lack of triacylglyccrols transfer activity affect both
chylomicrons1 formation in the intestine and VLDLs formation in the liver. This
protein associates lipids and apoproteins B, which arc present in chylomicrons,
VLDLs and LDLs. Low activity of MTP leads to abetalipoproteinemia, which
symptoms include absence of chylomicrons, VLDLs and LDLs in blood,
accumulation of lipid droplets in small intestinal cells, fat malabsorption,
steatorrhea, weight loss, deficiencies of fat-soluble vitamins and essential fatty
acids, retardation, ataxia.
Changes in relative concent rations of lipoproteins in blood arc analyzed for
diagnosis of many diseases. As lipoproteins arc distinguished by their density and
total charge, two methods arc used for their determination: ul trace nt rifugalion and
electrophoresis (Fig. 7.6).
320 Chapter 7. Lipid metaD ol ism

A B

Cm

LDL{p)

VLDL(pre-|ij

HDL(a)

Increase of density

Fig 7.6. Analysis ol serum mood lipoproteins by ultracentrifugation (A) and Uy electrophoresis (B).
A (a) — 12 hours after a meal; A (D) — 1 nour after a fatty meal; B — electrophoresis of blood
lipoproteins in fed state ifatty meal); CM — cnyto microns; pre-p !i poproteins (VLDLI, p lipoproteins
(LDL); ci lipoproteins (HOL)

Review tests
I. Lipoproteins compounds. Match She figure with the letter.

A. Cholesterol.
B. Integral apopop rolcins (apo B-100 or apo B-48).
C. Phospholipids.
D. Peripheral apoproteins (ex. apoAL apoAII, apo EK
E. TAG.
7.3. Disorders of Li pi ds Digestion and Transport 321

2. Lipoproteins compounds. Match ibc figure with the Idler

A. Cholesterol.
B. Integra! apoproteins (apoB-l00orapoB-48).
C. Phospholipids.
D. Peripheral apoproteins (ck. apo A-1, apoa-ILapoE).
E. Cholesterol esters.
J. Lipoproteins of human serum blood 2 hours
after a meal. Match the figure with the letter
(u Itracentrifuga t ton method). Lipoproteins
A. HDL density
B. LDL. increse
C. CM
D. VLDL
E. CM nascent.
4. Lipoproteins of human serum blood I4 hours
after a meal. Match the figure with the letter
(u El raceulrtfuga I ion metho d). Lipoproteins
A. CM. density
B. VLDL. increse
C. HDL
D LDL
E. CM nascent.

Situational Problems
1. A 50-years-old man consumes daily about 90 g of fat of animal origin (fatly
meat, butler, cheese), another man of the same age — 50 g offal of animal origin
and 30 g of plant oil. Which food: is healthier? For the answer:
a) name the differences in fatty acid composition of these TAGs:
b) draw the structures of the TAG molecules specific to animal fai solid at room
temperature and to olive oil liquid at the same temperature;
c) explain which TAGs arc more useful.
2. During the meal, two different persons both got some dietary lipids, but the
content of tally acids was different. First one goL the lipids containing oleic,
linolenic, linoleic, arachidonic, palmitoleic fatty acids (named in order of
its decreasing content in the fat). The latter one got the lipids containing
322 Chapter 7. Lipid metaD ol ism

palmitoleic, stearic, oleic and linoleic acids. Which person got a fat with the
higher nutritional value? To answer the question:
a) describe all possible ways of fatty acids structure coding; give the examples;
b) list all the essential components of a lipid nature;
c) explain, which lai from those two, would be more beneficial for nutritional
health, and why;
d) name the fatty acids — the precursors of biologically active compounds.
3. Clinicians work on the case of a patient with chronic pancreatitis. This disease
leads to insufficient secretion of the pancreatic juice into the small intestine.
What changes in the dietary lipid processing that disease will cause? For the
answer:
a) draw the reactions of TAG digestion and the conditions they require;
b) list all possible consequences of lipid i ndigestion;
c) list the essential lipid compounds that would have a faulty absorption due to
this disease;
d) name the other detrimental causes of lipid indigestion and malabsorption.
4. A patient with food indigestion has been screened and diagnosed with
steatorrhea (the excretion of abnormal quantities of fat with the feces owing
Lo reduced absorption of fat by the intestine). What arc the causes and
consequences of the steatorrhea? For the answer:
a) draw the scheme of TAG digestion;
b) explain the role of the pancreas in dietary lipid digestion:
cl make a scheme of dietary’ TAG assimilation, up lo the step of chylomicron
formation;
d) list the dietary recommendation for that particular patient.
5. A patient with cholelithiasis (gallstone disease) has long-term failure of the
bile salts passage into the duodenum. The observation has shown an increased
clotting lime. What are the possible causes of that symptom? For the answer;
a) name the essential nutrients that could be at a deficit in that patient;
b) explain the possible causes of the bleeding disorder;
ci name the other symptoms that can develop in that patient.
6. Scrum blood of a patient with dislipoproteinemia type 1 has milky appearance
even in fasting. If serum stays at low temperature (4’) for several hours, the fatly
layer appears on its surface. What arc the possible causes of these symptoms?
Answer the questions and do the following tasks:
a) what compounds of scrum blood must be tested in. the patient, in a
biochemical lab?
b) what is the possible diagnosis of the disease?
c) draw the reaction which docs not occur property in the patient’s blood:
d) explain how the products of the previous reaction arc used in adipose tissue
and heart in a healthy person I hour after meal.
7.. Heparin is a complex polysaccharide, a component of proteoglycans. Isolated
heparin used as an anticoagulant, binds to the protein antithrombin and inhibits
clot formation. As lipoprotein lipase is bound to the capillary endothelium
through proteoglycan, heparin also can bind to LP lipase and dislodge it from the
7.4. Synthesis of Fatty Acids ana Triacylglycerols — Upogenesis, Hormonal Regulation. Obesity 323

capillary wall. Why such treatment results in increased levels of iriacylglyccrols

in blood? For the answer:
a) draw the scheme representing the function of LP — lipase;
b) indicate the activator of this enzyme;
c) explain the mechanism of hypcnriacylglyccrolemia development in the
patient.
8. A 50-ycar-old woman, makes complain about persistent pain in a postprandial
right upper quadrant, which has been happening during the last few years.
Recently; the patient’s vision got worsen too, when it is dark. What arc the
molecular mechanisms of the sc two connected pathologies in that patient — the
gallbladder malfunction and the vision drop?
9. Obesity becomes epidemic in many countries due to excessive food consumption
and reduced physical activity The problem is that obesity has significant
health implications. Therefore, interest in weight reduction is widespread.
Orlistat, a modern anti-obesity drug, is a nonhyd roly zablc structural analog
of a triacylglyccrol and powerful inhibitor of the enzy me required for digestion
of TAG. Orlistat not only decreases fat absorption, but also stimulates the
production of the YY peptide, which gives a satiety sensation, and slows down
a food motion along the gastrointestinal tract. Explain the mechanisms of
Orlistat’s action on the weight loss. For that answer the questions and do the
following tasks:
a) what enzyme is being inhibited by Orlistat?
b) draw the reaction, which happens in the small intestinal, where this drug
works as an. inhibitor;
c) what type of inhibition has Orlistat as it is the structural analog of a
triacyl glycerol?
d) describe the mechanism of weight loss after orlistat treatment.

7.4. SYNTHESIS OF FATTY ACIDS AND TRI ACYLGLYCEROLS -

LIPOGENESIS, HORMONAL REGULATION. OBESITY
Fatty acids produced in the cells or obtained from the diet, arc used for the
triacy[glycerol synthesis — the major storage form of fuels in humans. Also, fatty
acids arc used for the synthesis of phospholipids — the major components of cell
membranes.
Fatty acids are synthesized mainly in the liver in a fed state, when blood levels of
glucose and insulin arc elevated. As an excess of dietary carbohydrates is consumed,
prod uctsofglucosc catabolism a re convened into fatty acidsand then to triacylglyccrols.
The substrates required for fatty acids synthesis are the products of glycolysis and
pentose phosphate pathway: acetyl-CoA, ATP, and NADPH. Reactions of glycolysis
convert glucose to pyruvate in the cytosol. Pyruvate enters mitochondrion and is
converted to acetyl-CoA by pyruvate dehydrogenase and to oxaloacctatc by pyruvate
carboxylase (Fig. 7.7). High levels of NADH and ATP in mitochondria reduce the rate
of the TCA cycle in the fed state inhibiting the regulatory enzymes. Asa result, acetyl-
CoA accumulates and condenses with oxaloacctatc to form citrate. This regulation
allows the continuous synthesis of citrate, which is transported from mitochondrion
to the cytosol. Citrate is cleaved: by citrate lyase to acetyl-CoA and oxaloacctatc in the
324 Chapter 7. Lipid metaD ol ism

cytosol Acetyl-CoA becomes available for fatty acids synthesis as the initial precursor.
Next reactions of fatty acids synthesis occur in the cytosol.

Fig. 7.7. Conversion of glucose catabolism products into palmitate me main tatty acid synthesized
in humans. Acetyl-CoA is transferred from mitochondrion to me cytosol in form of citrate. Then, the
released acetyt-CoA becomes the initial precursor of palmitic acid synthesis. The whole process is
stimulated Dy insulin inducing the synthesis of enzymes involved in conversion of glucose catabolism
products in fatty acids. T— The inducible enzymes: 1 — glucose 6-pnosphate dehydrogenase (the
enzyme of pentose pnospnaie pathway}; 2 — malic enzyme; 3 — acetyl-CoA carboxylase; 4 — fatty
acid synthase complex; 5 — citrate lyase

The first reaction of fatty acids synthesis is the conversion of acetyl-CoA to

malonyl-CoA by acetyl-CoA carboxylase:
v
o o
Acetyl-CoA carboxylase
CHa—’ C - SCO A + CO2 + ATP -------- —-------- — ------- * "OOC—CH2—C - SCoA + ADP + P,
Biotin
[ Acetyl-CoA) [Malonyl-CoA)

Acetyl-CoA carboxylase adds a carboxyl group to acetyl-CoA to form malonyi-

CoA. The enzymic belongs to ligases, requires ATP as a source of energy and biotin
as coenzyme. Acetyl-CoA carboxylase is the rate-limiting enzyme of the fatly acids
synthesis.
All I other reactions of the fatty acid synthesis arc catalyzed by the fatty acid synthase
complex. This complex is a large enzyme composed of two identical subunits (Fig. 7.8).
Each of them contains seven domains with specific catalytic activity and acyl carrier
protein (ACP). transferring the growing acyl chain from one catalytic site to another
(Fig. 7.9). Fatly acids synthesis is the cyclic pathway; malonyl-CoA serves as the donor
of two carbon atoms that arc added to the growing fatty acyl chain to form palmitate —
7.4. Synthesis of Fatty Acids ana Triacylglycerols — Upogenesis, Hormonal Regulation. ODesity 325

the end product. Palmitic acid is the main fatty acid synthesized in humans. Thus. the
previous name of fatly acid synthase was palmitate synthase.

Fig. 7.8. The general structure of the tatty acid synthase complex: 1 — SH cysteinyl sulfhydryl group
to which acetyl is attached; 2 — SH phospnopafitetneinyi group to which maionyt is attached

Fig. 7.9. The active sites of me fatty acid synthase complex. ACP — acyl carrier protein, Cys —
SH — radical of cysteine

Al first, an acetyl and malonyl arc transferred from acetyl-Co A and malonyl-CoA
to the SH-groups of fatty acid synthase complex by acetyl and malonyl transferases
(Fig. 7. IO, I, 2). Next, acetyl and malonyl moictics condense with the release of the
malonyl carboxyl group as CO.. A four-carbon (3-keto butyryl is formed (3). In the next
three mac l ions, p-keto group is reduced to hydroxyl group by NAD PH, then the water
is removed to form double bond, which is reduced by NADPH (4.5,6). In the result of
the first cycle, the original acetyl group is elongated by two carbons. This sequence of
reactions is repeated until the chain becomes 16 carbons in length. In total, 7 cycles arc
required for palmitoyl to being synthci ized. Then, palmitate is released from the fatty
acid synthase by reaction of hydrolyzis. The summary’ equation of pal mi Late synthesis is:

Acetyl-CoA + 7 malonyl-CoA + I4NADPH + 14 H ->Cl.Hs|COOH 4- 7 CO. +

8 CoASH + 14 NADP 4- 6 H2O.
326 Chapter 7. Lipid metaD ol ism

PaJmitoyl-E

Palmitate

Fi^TTO. Synthesis of palmitate by me fatty acid synthase complex. 1 - Transfer of acetyl from acetyl-
CoA to SH group of fatty acid synthase. 2 - Transfer of malonyl from malonyl-CoA to SH group of
me enzyme. 3 — Condensation of acetyl and malonyl groups wim the release of the malonyl carboxyl
group as CO?. 4 — Reduction of the P-keta group to hydroxyl group. 5 — Dehydration. 6 - Reduction
of the double bond. 7 — Buteryi is transffered to SH group of site ®. Then the cycle repeats until the
chain becomes 16 carbons in length and palmitate is released by reaction of hydrolyzis

Two carbon atoms — l6Lhand 15th of the palmitate arc derived front an acetyl-CoA.
The other carbons in the aliphatic chain arc derived from malonyl-CoA.
7.4. Synthesis of Fatty Acids ana Triacyigiycerois — Up agenesis, Hormonal Regulation. Obesity 327

The synthesized palmitate is used for the synthesis of other lipids, mainly
triacy(glycerols and glyccrophospholipids, or can be elongated by a series of reactions
in the endoplasmic reticulum. These reactions start from conversion of palmitate
(C 16:0) to palmitoyl-Co A, which is elongated by malonyl-Co A to stcaryl-CoA
(C 18:0). Very long-chain fatty acids (C22 to C24) can also be synthesized this way,
particularly in the brain. Desaturation of synthesized saturated fatty acids is possible,
but the main products arc palmitoleic (16:1 A9) and oleic (18:1 A9) acids (Fig. 7.11).
Oxygen (OjX cytochrome b5 and NADU arc required for desaturation reactions.
Polyunsaturated fatty acids of series tn 6 like linoleic (18:2 A 9,12 w6) and like
a-linolenic (18:3 A 9,12,15 co3) acids arc not synthesized in mammals as they lack of
enzymes necessary to insert double bonds between the 9th carbon and os-carbon of
fatty acids. Humans can obtain them only from dietary plant or fish oils (essential fatty
acids). In humans, linoleic and a-linolcnic acidscan be elongated to arachidonic and
eicosapentacnoic acids, which are the precursors for the synthesis of prostaglandins
and other eicosanoids.

Fig. 7.11. Synthesis of unsaturated fatty acids

The regulation of fatty acids synthesis. The hormone activating fatty acid synthesis
is insulin. Insulin, stimulates both glycolysis and pentose phosphate pathway of glucose
catabolism, acting through their regulatory enzymes bv increasing their activity and
synthesis (Fig. 7.7). This regulation makes the substrates for fatly acid synthesis more
available. Insulin, also induces the synthesis of acetyl-CoA carboxylase — the rate
limiting enzyme of fatty acids synthesis and regulates its activity.
The activity of the enzyme is regulated by:
► phosphoryiation/dephosphorylation;
► allosteric modification;
► induct ion/repression of its synthesis.
in fed state. acctyl-CoA carboxylase is dephospho rydated by in.su I in-stimulated
phosphatase (Fig. 7.12). The individual dcphosphorylatcd molecules of acetyl-
CoA carboxylase, consisting of four subunits, are polymerized and become active.
Polymerization is stimulated by citrate allostcrically activating the process. The level
of citrate is elevated in fed state in cytosol of hepatocytes and so the synthesis of fatty
328 Chapter 7. Lipid metaD ol ism

acids. During fasting, acetyl-CoA carboxylase becomes inactive and the synthesis of
fatty acids is inhibited. High secretion of glucagon or epinephrine (fasting or physical
activity) causes phosphorylation and inhibition of acelyI-CoA. carboxylase by protein
kinase A. Acetyl-CoA carboxylase is also phosphorylated by AMP-activated protein
kinase and becomes inactive. AM P-acrivaled protein kinase is activated in low energy
level of the cells. AMP is the product of ATP degradation and the most sensitive
indicator of low energy levels in the cells. This regulation helps the cell to survive when
the energy level is very tow by switching off noncssential biosynthetic pathways like
fatty acids synthesis.

4
Glucagon, Epinephrine

Fig. 7.1 Z Regulation of me acetyl-CoA carDoxytase activity

Palmitoyl-CoA, formed from palmitate, the end product of fatty acid synthesis,
inhibits acetyl-CoA. carboxylase, through depolymerization. This is feedback
inhibition of fatly acid synthesis by the high level of the end product.
Acetyl-CoA carboxylase synthesis is also regulated by insulin at the level of
transcription. An excess food consumption causes the over secretion of insulin, so the
rate of transcription of the gene coding acetyl-CoA carboxylase increases. Low-calorie
diet (low insulin secretion) leads to the suppression of the transcription of this gene.
Triacylgliycerol synthesis occurs in the liver and adipose tissue and requires glycerol
3-phosphate and fatty acids as substrates. The origination of glycerol 3-phospha to,
which provides glycerol for triacylglyccrol synthesis, and fatty acids required for
that arc different in these two tissues. In the liver, glycerol 3-phosphate is produced
from the reduction of dihydroxyacctone phosphate derived from glycolysis or by the
phosphorylation of glycerol delivered by blood (Fig. 7.I3). The phosphorylation of
glycerol, is catalyzed by glycerol kinase only in the liver. But adipose tissue lacks the last
enzyme, therefore, dihydroxyacctone phosphate can be produced only from glycolysis.
7.4. Synthesis of Fatty Acids ana Triacylglycerols — Upogenesis, Hormonal Regulation. ODesity 329

Liver and adipose tissue

Liver
Giuc-ose

Gtvcolysis *

I
CHnOH
I
C—O O CHpOH
I
CHj—O—p — 0“ CHOH

CHsOH
D^rydroxyacelcne
[ijlyceroiJ
ph os prate

NADH *- H+-^ ATP

Glycerol 3-phosphate1 ' Glycerol

CHj tJ

[L-Xjlycera* 3-phoaphatej

2 R-COCoA I
Acyl transferase I
2 Go ASH-Xj
0
I Liver
Crtj—0 —C—R|

i Attachment di head
ch—o—c —b2 Dup (serine, choline,
elhanolamine, etc.)

Ri
I
Ch?— o—P — o-
? Q
I
CHn" Q — C ■— R |
I O"
I ?
[Phosphatidic dzid CH-O— C”Hn
I
Gh^O h I ?
[1,2-Diacylgfycerolj CHn— O— P — O

________ 0-________
, erophosphc^pid
Acyl transferase

I
CHt— O“ c— r,
I
Ch—O—C^R2
?
I O
I
Cbt^o—c — r3
[ Triacylglycerol]

Fig. 713. Synthesis of triacylglycerols in the liver and adipose tissue

330 Chapter 7. Lipid metaD ol ism

In the liver, fatty acids synthesized de now from products of glucose catabolism arc
used for triacylglyccrols synthesis (Fig. 7.14). Two fatty acyl-CoA react with glycerol
3-phosphate to form phosphatidic acid and then TAG.

'Malonyl-CSA|

Phcsphat die acic ;

Fitp 7.14. CoirverSion of glucose into fatty acids and triacylgtycerols in the liver in fed state

In adipose tissue, the fatty acids required for the TAG synthesis mainly originate
from reaction catalyzed by LP lipase (Fig. 7.15). Lipoprotein lipase attached to the
capillary endothelial cells recognizes apo CH on the surface of VLDL as it docs with
chylomicron. This enzyme hydrolyzes triacylglyccrols in VLDL and chylomicrons to
glycerol and fatly acids. Fatty acid enters the adipose cells and is converted to acyl-
CoA. Then two acyl-CoA react with glycerol 3-phosphate to form phosphatidic acid.
Dcphosphorylation of phosphatidic acid produces diacylglyccrol. Finally, the third
fatty acyl-CoA reacts with diacylglyccrol to form triacylglycerol.
TAG synthesized in. the adipocytes is the main storage of fuels in human body. TAG
synthesized in the liver (endogenous fats) are packaged into the VLD L and delivered
io other tissues. The conversion of glucose to fatty acids and TAG in adipose cells is
possible, although the liver is the major site of fatty acid and TAG synthesis.
7.4. Synthesis of Fatty Acids ana Triacylglycerols — Upogenesis, Hormonal Regulation. Obesity 331

Fig. 7.15. Conversion of the fatty acids, delivered as TAG of chylomicrons and VLDL to Iriacylglycerols
stored in adipose tissue, insulin stimulates the synthesis and secretion of LP — tipase from adipose
cells to capillary walls and activates GLUT — 4 in plasma membranes, allowing glucose to enter the
cells. Thus, the synthesis of triacylgiycer-ots increases in the adipose ceils in fed state. FA — fatty
acids, OHAR — dihydroxyacetone phosphate

Transport of endogenous TAG from the liver to the peripheral tissues. The
triacylglycerols synthesized in cytosol of the hepatocytes, arc packaged into the
VLDL — ver? low density lipoprotein, together with phospholipids, cholesterol, and
proteins. The structure of VLDL particle is similar lo the structure of chylomicron
(Fig. 7.4). The major apoprotein of VLDL is apo B-100 wound through the surface
of VLDL particle. MTP — the microsomal triglyceride transfer protein, required for
chylomicron formation, is also required for VLDL formation. VLDL, formed! in the
liver as nascent panicles, arc secreted lo blood where they acquire apoC i I and apoE
from HDL to become mature VLDL particles (Fig. 7.I6). The function of VLDL,
containing 55% of triacylglycerols, is to deliver triacylglycerols synthesized in the liver
to other tissues, particular to adipose tissue and muscles.
In blood, LP lipase hydrolyzes triacylglycerols in VLDL and the content of
TAG in Lhe particles decreases. The released fatty acids arc transported lo the cells
mainly to adipocytesand hepatocytes. Glycerol is delivered to the liver. Thus, VLDL
becomes al first IDL (intermediate density lipoproteins), and then LDL (low density
lipoproteins). LDLs contain only abouL 7%- of TAG. The major compounds of LDL
arc cholesterol and cholesterol esters (Fig. 7.I6, Table 7.3). In Lhe liver TAG of VLDL
and chylomicrons arc also cleaved by hepatic triglyceride lipase (HTGL) which is also
attached to the capillary walls. The subsumes of this enzyme are lipoproteins already
partial!v digested by LP lipase. The hepatic triglyceride lipase facilitates conversion of
IDL into LDL
332 Chapter 7. Lipid metaD ol ism

Liver

(vlDl nascent j
(55% TAG, 15% C + CE)

ApoE

[ VLDL mature^

LP -Lipase

3 RCOOH
Glycerol

LP -Lipase

3 RCOOH
Glycerol

(7% TAG, 50% C + CE)

fig. 7.16. Metabolism of VLDL in blood HOL donates apoCli and apuE to nascent VLDL converting
inem to mature VLDL. Then LP lipase hydrolyzes TAG in VLDL. The amount of TAG in the particles
decreases and they become at first IDL (intermediate density lipoproteins} and then LDL (low density
lipoproteins}. C + CE cholesterol and cholesterol esters

[n case of LP lipase deficiency, the levels of VLDLs arc elevated as well as levels
of chylomicrons and TAG, and dislipoproteinemia type 1 develops. Patients with this
disease have exceedingly high plasma triacylglycerol concentration (over l()00mg/dl,
normal range <160 mg/dl). These patients suffer from eruptive xanthomas (yellowish
triacylglycerol deposits in the skin) and chronic pancreatitis.
Obesity. As the fatty acids in tri acyl glycerols of adipose tissue conic both from
chylomicrons and VLDL, humans produce fat stores both, from dietary fat delivered
by chylomicrons and dietary carbohydrates, which produce fat of VLDL. Therefore,
overeat i ng fat, carbohydrates and even proteins leads to excess accumulation of fat and
obesity.
Recent investigations have shown that the adipose tissue is not only the storage
of triacylglycerols but also is active endocrine organ secreting different hormones
and other regulatory factors (Fig. 7.17). The secretion of these regulatory factors
significantly changes in case of obesity. One of the first discovered factors was leptin —
the peptide released from adipocytes as their fat accumulation increases. Leptin, binds
to receptors in hypothalamus and stimulates the release of neuropeptide V into blood
that signal the cessation of eating. I?ut in some obese patients leptin, receptors become
less sensitive to leptin that leads to dysregulaiion of appetite and weight gain. Leptin
7.4. Synthesis of Fatty Acids ana Triacyigiycerois — Upogenesis, Hormonal Regulation. Odesity 333

is coded by Mutations in this gene lead to obesity as leptin is the

hormone regulating the mass of adipose tissue.

Adiponectin LJP-Lipase
Angiotensin ll

Eicosanoids

FFA

IL - 6

Fig. 7.17. Secretion of honnooes and other Biologically active molecules By adipocytes. Leptin and
resistin secretion increase in case of obesity that cause the cellular resistance io insulin. Tne level of
free fatty acids (FFA) in D loo a is elevated. Secretion of adiponectin which increases the sensitivity of
the tissues to insulin decreases with the accumulation of fat in me adipocytes. IL — 6 interleukin —
stimulates acute phase protein synthesis

Fatly acids secreted From adipocytes arc transported by blood and taken up by the
liver. Some of fatty acids entered the hepatocytes can regulate the gene expression.
Faliy acids bind to the special receptors named PPARs — peroxisome proliferator-
act hated receptors (sec Ch. 7.6). These receptors arc members of a nuclear receptor
family and, when, activated, regulate the rate of transcription. This mechanism
resembles the action of steroid hormones, which arc also hydrophobic molecules, but
the receptors of hormones arc more selective.
The development ofobesity is a major problem worldwide and is associated with the
failure of energy balance mechanisms and regulation of food intake. Clinical obesity is
now clearly defined in terms of height and weight through the body mass index (UM I),
which is calculated as the weight in kilograms divided by the height in meters:

Weight (kg)
BMl =----------------- .
Height (m)2

RM I 25-30 kg/m2 is classified as overweight or grade 1 obesity. I? Ml >30 kg/m2

is clinical or grade II obesity, and RM I >40 kg/m2 is classified as morbid or grade
III obesity. Medical complications of obesity arc widespread and arc associated with
both chronic and acute diseases. The most important of the associated diseases is type
2 diabetes mdlitus: 80% of the patient with this type of diabetes have obesity. Obesity
associated illnesses include coronary heart disease, hypertension, stroke, arthritis, and
334 Chapter 7. Lipid metaD ol ism

some forms of cancer. As obesity is the primary risk for so many diseases, it has to be
treated intensively.

The normal quantitative characteristics of the TAG and fatty acids metabolism.
Blood:
TAG < 160 mg/dl:
Free fatty acids 1.0-25 nig/dl.

Review Test
1. Fatly acid synthesis. Match the figure with the letter:

HS

A. NAD PH.
B. Malonyl-CoA.
C. Acetvl-CoA.
D. NADP4.
7.4. Synthesis of Fatty Acids ana Triacylglycerols — Upogenesis, Hormonal Regulation. ODesity 335

2. Regulation of the key enzyme of the fatty acid synthesis. Match the figure with the letter:

(p

A. Cortisol.
B. Glucagon.
C. Phosphatase.
D. Pyruvate.
E. Insulin.

3. TAG synthesis from products of glucose ncthabolism. Match the figure with the letter:

[Glucogej

CHa-O— C— Ft 3 CHn—QH
Triacylgiycerol ] C3}
336 Chapter 7. Lipid metaD ol ism

A. TAG.
B. DAG.
C. Glycerol 3-phosphaic.
D. Dihydroxyacetonc phosphate.
E. Phosphatidic acid.
4. Conversion of glucose in fatly acids and Iriacylglycerok in the liver in fed state. Match
the figure with the letter:

H.3PO4

A. Malonyl-CoA.
B. Phosphatidic acid.
C. Glycerol-3P.
D. Monoacylglyccrol.
7.4. Synthesis of Fatty Acids artel Triacyigiycerois — Upogenesis, Hormonal Regulation. Obesity 337

Situational Problems
I . What are the numbers of carbon atoms derived from the first acelyl-CoA that
binds to fatly acid synthase complex in palmitate, the end product ofthe pathway?
For the answer:
a) name the initial substrates for fatty acid synthesis and their source;
b) draw die reactions of the first spiral of fatly acid synthesis;
b) mark the carbon atoms (*> derived from the first acc-tyl-CoA in the product of
the first spiral of fatty acid synthesis;
c) answer the question of the problem.
2. After gelling 300 g of carbohydrates with her meal, a student went to bed. What
metabolic pathways of fatty acids have been activated in the liver about an. hour
after having a meal? For the answer:
a) describe the glucose metabolism in the liver in this condition;
b) name the metabolic pathway of fatty acids which, has been activated:
c) draw the scheme of this pathway and explain the fate of the end products of
the pathway in the liver;
d) explain the regulation of the pathway.
3. A patient has got excess carbohydrate meal for the years and gain the weight. To
explain this:
a) draw the schemes of TAG synthesis in the liver;
b) describe the transport of TAG from the liver to adipose tissue;
c) describe the functions of insulin in the conversion of glucose to TAG in the
liver and adipose tissue.
4. Glucose containing l4C atoms was added to isolated hepatocytes in an experiment.
If the glucose was added in excess, the rale of triacylglyccrol synthesis increased:.
The determination of carbon atoms in the triacylglycerol molecule confirmed
that all carbon atoms in glycerol and fatty acids were 14C atoms. Explain the
results of the experiment. For that:
a) name the metabolites required for triacylglycerol synthesis, which arc
produced by glucose catabolism:
bldraw the scheme of glucose catabolism to metabolites required for
triacylglyccrol synthesis which would explain the appearance of “C atoms in
the triacylglyccrol molecule:
c)draw the scheme representing the conversion of extra glucose to
triacy I glycerols in. the liver.
5. One student has got 300 g of carbohydrates and 50 g of proteins with dinner,
another — 300 g of carbohydrates and 50 g of fats. Both d id not carry out any
physical activity. Explain the difference in the lipoprotein composition in their
blood one hour after the meal. For the answer:
a) name all types of lipoproteins and describe their structure and functions:
bldraw. the schemes explaining the difference in the lipoprotein formation in
both students:
c) name the methods used for lipoproteins analysis of scrum blood;
d) draw the scheme of electrophoresis of sc rum lipoproteins for both students.
338 Chapter 7. Lipid metaD ol ism

6. Kwashiorkor is the disease caused by a deficiency of proteins in the diet that

is adequate in calorics due to carbohydrates. Children with kwashiorkor suffer
from muscle wasting, low level of plasma proteins and fatty liver. Explain the
cause of increased content of TAG in the liver (fatty liver). Answer the following
questions and do the task:
a) which lipoproteins transport fats the from liver to peripheral tissues?
b) which compounds arc present in the shell and inside of the lipoproteins, write
the general scheme of VLDL structure;
c) name the apoproteins required for VLDL formation;
d) explain the possible cause of fatly liver development.
7. During several days. Patient A got a meal with high caloric value, and Patient
11 — with, a low caloric value. What arc the differences in the lipid metabolism
in these two patients? What is the insulin/glucagon ratio in these patients after
a meal? Which metabolic pathways rather activated in Patient A and how is it
being controlled?
8. The adipose tissue is not only store triacylglyccrols but also is active endocrine
organ. Explain this function of the adipose tissue. For that:
a) explain the ^clinical obesity* and possible consequences of the disease;
b) name the biologically active molecules secreted by the adipose tissue;
c) explain how secretion of these molecules changes with development of
obesity.
9. A daily diet of a 5 5-year-old woman, consisted of a 500 g of carbohydrates, 100 g
of animal fats and 150 g of proteins, at the background of low physical activity.
What possible consequences for the lipid metabolism such lifestyle could trigger?
For the answer:
a) compare the daily food consumption of the patient with the norm for this age
and level of physical activity;
b) draw the scheme of TAG synthesis in the liver and continue writh their
following transport to the tissues;
c) draw the scheme of metabolic pathways which arc more active in adipocytes
of this patient compared with an individual of the same age consuming
balanced diet;
d) name the biologically active molecules released by adipocytes of th is pa tic nt.
10. Persistent waste with toxic substances, for example, an alcohol or some
medications, can cause fatty liver disease. What arc the possible mechanisms
lor that condition? What arc the reasons for using methionine amino acid for
several liver problems, specifically as a substance lowering the risk of developing
fatty liver disease?

75. MOBILIZATION OF STORED FATS FROM ADIPOSE TISSUE

Triacylglycerols stored in adipose tissue arc used during fasting, intensive physical
activity and stress as source of energy. The mobilization of fats from the adipocytes is
stimulated by glucagon, epinephrine and neurotransmitter norepinephrine released
by sympathetic nerve system. The hormones stimulate lipolysis through the cyclic
7.5. Mobilizaiion of Stored Fats from Adipose Tissue 339

AM P system, rising the cAMP level in the adipocytes (Fig. 7.18). Protein kinase A is
activated by cAMP and phosphorylates hormone sensitive lipase (HSL), also called
adipose triacylglyccrol lipase. This enzyme cleaves a fatty acid from a triacyl glycerol.
Then other lipases complete hydrolyzis to glycerol and fatty acids, which are released
into the bloodstream. Fatty acids, as water insoluble molecules, bind to the major
serum, protein albumin and are transported to muscles and other tissues, where; they
are oxidized to CO, and water to produce energy. Glycerol travels to the liver and
serves as a substrate of gluconeogenesis during prolonged fasting.

Fi&7.18. Mobilization ol stored fats from adipose tissue. R — receptor: G — G-protein; AC — aaenyiate
cyclase

Recently, an important mechanism regulating the release of fatty acids from the
adipocytes has been described. It has been revealed that up to 40% of fatty acids
released from lipolysis arc rc-cstcrificd to triacvlglyccrols in the adipocytes. The
glycerol-3 phosphate required for triacylglycerol rosy n thesis in adipocytes is formed
by the shortened pathway of gluconeogenesis. The main substrates of gluconeogenesis
arc lactate and amino acids which arc taken from the blood. This mechanism controls
the amount of fatty acids, leaving the adipocytes during prolonged fasting.
340 Chapter 7. Lipid metaD ol ism

ZL UTILIZATION OF FATTY ACIDS FOR ENERGY PRODUCTION -

^OXIDATION. DISORDERS OF ^OXIDATION. THE OTHER PATHWAYS
OF FATTY ACIDS OXIDATION
Most tissues can use fatty acids as fuel. Fatty acids arc major energy source in
cardiac and skeletal muscles, but the brain oxidizes fatty acids very poorly, because of
the limited transport across the blood-brain barrier. Erythrocytes cannot oxidize fatty
acids as they do not have mitochondria, the site of fatty acids oxidation.
The main fatly acids released from adipose tissue and delivered to the other tissues
contain (12—18 carbons). These acids travel with the blood bound to albumin. They
enter the cells by diffusion through the plasma membrane and by facilitated transport
with a fatty acid-binding protein in the plasma membrane. After entering the cell a
fatty acid must be activated to acyl-Co A derivative before it can participate in any
metabolic pathway. The reaction requires the enzyme acyl-CoA synthetase, ATPt
CoASH (sec 7.2). Then acyl-CoAs are oxidized in the [i-oxidation pathway which is
integrated with electron transport chain (ETC) and the tricarboxylic acid cycle (TCA).
fj-oxidation is the specific aerobic pathway of fatty acids oxidation which occur in
mitochondria matrix. The pathway is the spiral process and in every spiral two-carbon
fragments — acetyl-CoA — is removed sequentially from the carboxyl end of the fatty
acid. Therefore, the end product of the pathway isacctyl-CoA.
The fatty acids arc converted al first to their active-CoA derivatives in. cytoplasm,
but they arc oxidized in the matrix of the mitochondrion. The inner membrane of
mitochondrion is impermeable to CoA and its derivatives so fatty acvls are transported
into mitochondria using the special carrier — carnitine (Fig. 7.19). The acyl group is
transferred from CoA to carnitine by carnitine palmitoyltransferase I (CPT I) on the
outer mitochondrial membrane:

+ N (CH3)s

CHg- (CHaJn—C SCOA + HO -CH- CH2 COOH

Carnitine il

Carnitine palmitoyl
transferase I (CPT I)

O ch2
CH
I 1
CHa—(CH^“C—O —CH—CH£—COOH + CoASH

[Acylcarnitine]
7.6. Utilization uf Fatly Acids For Energy Production - ^-oxidation. Disorders of p -oxidation... 341

Acylcarnilinc and free carnitine arc then exchanged across the inner mitochondrial
membrane by carnitine aeylcarnitinc translocase. Finally, the fatty acyl group is
transferred back to CoA by carnitine palmitoyltransferase II (CPT 11) on the matrix
side of the inner membrane. This process functions primarily in the transport of fatly
aeyl-CoAswith 12-18 carbons. Entry of shorter chain fatty acids into the mitochondria
is independent of the carnitine; they cross the inner mitochondrial membrane and arc
converted to CoA derivatives in the matrix.

Fatty acids Acyl-CoA CoASH

Outer
mitochondrial
membrane

inner
mitochondrial
membrane

Matrix
p-oxidation ■*— Acyl-CoA CoASH

CPT I — carnitine palmitoyltransferase

Fig. 7.19. Transport of long-chain fatty acids into mitoctioisdriaiL
I isthe rate-limiting enzyme of me p-oxidation pathway. The activity of me enzyme determines the
supply of substrate for p-oxination in mitochondrial matrix. Mafonyt-CoA synthesized Dy acetyl-CoA
carboxylase in fed state, inhibits CPT 1 and decreases me rale of fatty acids oxidation

Carnitine palmitoyltransferase I (CPT 1) is the rate-limiting enzyme of the

p-oxidation pathway because its rate controls the entry of fatty acids into the
mitochondria and therefore determines the supply of substrate for p-oxidation in
mitochondrial matrix.
The sequence of four reactions of the (1-oxidalion cycle is presented in Fig. 7.20.
Once a fatty acyl-CoA is formed in the mitochondrial matrix, it can be oxidized by
FAD-depended acyl-CoA dehydrogenase. The products arc the enoyl-CoA with
double bond between a and ft carbon atoms of acyl and FA DH? transferring electrons
to coenzyme Q of electron transport chain. In the next step, water molecule is added
by enoyl-CoA hydratase to the double bond. An -OH from waler is added to the p
carbon and -H from water molecule is added to the a carbon. The product of the
reaction is p-hydroxy acyl-CoA. At the third step of p-oxidalion. the hydroxyl group
on the p carbon is oxidized to a ketone by a p-hydroxy acyl-CoA dehydrogenase: with
the formation of p-kctoacyl-CoA. In this reaction, the electrons arc transferred to
NAD to form N.ADH. NADH is reoxidized by ETC. producing ATP. In the last
342 Chapter 7. Lipid metaD ol ism

reaction of she sequence, the bond between a- and [3-carbons is cleaved by an enzyme
P-kelolhiolasc. The products are the acetyl-CoA and the fatty acyl-CoA that is two
carbons shorter than the original The shortened fatly acyl-CoA repeats these four
steps until all of its carbons are converted to acetyl-CoA. In the last spiral, cleavage
of the four-carbon acyl-CoA produces two acetyl-CoA. Thus, an even-chain fatly
acid such as palmitoyl-CoA, which has 16 carbons, repeats the 7 spiralsand produces
8 aceiyi-CoA, 7 FADH2 and 7 NAD FL

O
fi a
CH3W CH2“jCH2- - CHq- C~SCoA Acyl-CoA
4
Cn

Acyf-CoA
dehydrogenase
2 ATP

Enoyl-CoA

|^-h2o
Enoyl-CoA hydratase

.Q OH O
—
d
CH3wCH2—CH—CH2 C p-hyd roxyacyl-CoA
£
9
NAD’
ETC fS-hydroxyacyt-CoA
NADH+H’’ —■ 3 ATP dehydrogenase

O
1
CHjwCH; CHs -C~SCoA p-Ketoacyl-CoA

CoASH
p-Ketothiotase

CH3v^CH?—

Fig. 720. P-Oxidation of fatty acids

7.6. Utilization of Fatly Acids For Energy Pr oduction - fi-oxidation. Disorders of p -oxidation.- 343

The complete oxidation of fatty acid up to CO. and H,0 includes p-oxidation
pathway, oxidation of acetyl-CoA in the TCA cycle and oxidation of FADH, and
NADH in ETC. The theoretical energy yield from the oxidation of I mol of palmitoyl-
CoA to 8 mol of acetyl-CoA is, therefore. 35 mol of ATP: 2 for each of the 7 FADH.,
and 3 for each of the 7 NADH (7x2 +7x3= 35 ATP). The oxidation of 8 mol of
acetyl-CoA in the TCA cycle produces 96 mol of ATP (8x12). Thus, the complete
oxidation of palmiloyl-CoA io CO2 and H,0 produces 131 mol of ATP. To calculate
the energy yield from I mol of palmitate, two ATP need to be subtracted from the total
because two high energy bonds are cleaved when palmitate is activated to palmiloyl-
CoA (35+96-2= 129). The real production of ATP from the oxidation of palmitate is
approximately 106 mol due to the dissipation of energy.
Each of the four reactions of p-oxidation is catalyzed by several different enzymes
that have specificity for fatty acids of different chain length. The long-chain fatly acyl-
CoA starts to oxidize by long-chain fatty acyl-CoA dehydrogenase (LOAD). As the
length of the acyl chain becomes shorter, it is transferred to the enzyme that acts on
shorter acyl chains. Medium-chain fatly acyl-Co A dehydrogenase (MCAD) and short
chain fatty acyi-CoA dehydrogenase continue to oxidize fatly acyl to acetyl-CoA.
Regulation of the p-oxidation. Fatty acids arc used as fuel mainly when they arc
released from adipose tissue in response to hormones that signal fasting (glucagon) or
physical activity (epinephrine). The process of p-oxidation is regulated by the cells1
requirements for energy (N.ADH/NAD and ATP/ADP ratio), as it depends on. the
rate of NADH and FADH2 rcoxidation in the electron transport chain. Another type
of regulation occurs through the key enzyme carnitine: palmitoyl transferase I (CRT I).
The activity of this rale-limiting enzyme determines the rale of fatty acids transport into
the mitochondrial matrix where p-oxidadon occurs. Carnitine: palmitoyltransferase I
is inhibited by malonyl-CoA synthesized by acetyl-CoA carboxylase. Malonyl-CoA
is the first metabolite in the pathway of fatty acids synthesis activated by insulin.
Thus, if fatty acids synthesis starts in. a fed' state, malonyl-CoA is formed and inhibits
the regulatory enzyme of P-oxidation and fatty acids arc not oxidized. Glucagon
and epinephrine inactivate the enzyme synthesizing malonyl-CoA — the inhibitor
of P-oxidation (Ch. 7.4), so in fasting or physical activity, the rate of p-oxidation
increases. Low levels of citrate and high levels of acyl-CoA also stimulate P-oxidation
by action on the acetyl-CoA carboxylase. As the rate of P-oxidation depends on
substrate availability, it increases when fatty acid levels are elevated in blood under the
action of glucagon or epinephrine.
Disorders of p-oxidation. Recently, medium-chain fatly acyl-CoA dehydrogenase
(MCAD) deficiency has emerged as one of the most common of the inborn diseases.
The frequency of MCAD deficiency is 1 in 15 OCHJ. In MCAD deficiency, fatty acids
arc oxidized only until they reach medium chain IcngLh. As a result, p-oxidation is
not completed, and ATP synthesis reduces. Therefore, the body must rely mainly on
oxidation of blood glucose to meet its energy needs. The most frequent manifestation
of MCAD deficiency is low levels of glucose and ketone bodies in the blood during
fasting (intermittent hypokciotic hypoglycemia). In healthy individuals, P-oxidation
is needed to produce acetyl-CoA for ketogenesis and ATP for gluconeogenesis during
fasting. In MCAD deficiency both pathways arc compromised, thus, hypoglycemia
344 Chapter 7. Lipid metaD ol ism

and low levels of ketone bodies in the blood develops. The combined deficiency of
ketone bodies and glucose is especially bad for the brain as it depends on a mix of
ketone bodies and glucose during prolonged fasting. The management of MCAD
deficient patients includes the intake of a relatively high carbohydrate diet and the
avoidance of prolog fasting (more than 6-7 hours for adults).
Another cause of fl-oxidation defects is carnitine deficiency. Il can be caused by
impaired carnitine synthesis in the kidneys and liver. Carnitine deficiency in skeletal
muscle causes muscle weakness and cramps on exertion.
Mitochondrial p-oxidation oxidizes unbranchcd saturated fatty acids with even
number of carbon atoms and a chain length up to 20 carbons, fatty acids with other
structures arc oxidized through some other metabolic pathways.
► Oxidation of unsaturated fatty acids requires modifications of their double bonds
before p-o.xidation. Their complete oxidation produces 2 ATP less for each
double bond then the saturated fatty acid with the same number of carbons.
* a-Oxidation is a minor pathway in the peroxisomes, mitochondria, and ER.
It oxidizes ct-carbon of fatty acids releasing CO3 and is needed for catabolism
of methylated fatty acids. In Refsum disease, a recessively inherited defect of
peroxisomal a-oxidation causes the accumulation of branched-chain fatty
acid - phytanic acid which originates from chlorophyll in green vegetables
and accumulates in the fat. meat and milk of ruminants. Refsum disease leads
to slowly progressive peripheral neuropathy and blindness. As phytanic acid
is obtained only from diet, placing patients on a low — phytanic acid diet has
resulted in marked improvement.
► u>-Oxidation is the oxidation at the co-carbon. the fatty acid (the terminal methyl
group} by the enzymes in the endoplasmic reticulum. These enzymes need
cytochrome P450, CL and NAD PH. Fatty acids arc oxidized to dicarboxy lie
acids with 6 to 10 carbons. Dicarboxylic acids undergo P-oxidation or excrete in
urine as water-soluble products.
► Peroxisomal p-oxidation. Peroxisomes arc present mainly in the liver and
contain the enzymes for the oxidation of very-long-chain fatty acids with length,
of 20 carbons or more. This pathway is similar to mitochondrial p-oxidation,
but in the very first reaction peroxide (H2O2) is produced instead of FAD PL.
The sequence of the other reactions remains the same, releasing the NADH and
acetyl-CoA. Peroxisomal catalase inactivates hydrogen peroxide by conversion
to water and O2. The octanoyl-CoA that, is the end product of peroxisomal
oxidation leaves the peroxisome and the octanoyt radical is transferred through
I he inner mitochondrial membrane by the medium chain-length acylcarnitinc
transferase. In the mitochondrion, it enters the P-oxidation pathway and then is
oxidizes to CO, and water.
The function of the peroxisomal a- and p-oxidation and microsomal m-oxidation
is to decrease levels of water- insoluble fatty acids or xenobioiic compounds with a fatly
acid like structure that would become toxic to cells al high concentration.
Peroxisome pro I iterator-activated receptors (PPARs) play an important role in
lipid metabolism in the liver. The PPARs arc members of a nuclear receptor family
and, when activated, stimulate the gene transcription, in the liver, PPARs regulate
7.7. Synthesis artel Oxidation of Ketone Bodies. Ketonemia, Ketoacidosis S45

the activity of the genes that arc involved in fatly acid intake and oxidation. There
arc three major PPAR isoforms a, p and y. The major form in the liver is a. Fatty
acids are the endogenous ligand for PPARa. When the levels of fatty acids in blood
and hepatocytes are increased, there is increased gene transcription, for the enzymes
involved in fatly acid oxidation. The drugs ftbrates bind to PPARs in the liver activate
them and improve lipid me La holism. The ftbrates arc prescribed for the patients with
elevated triacylglyccrols because they increase the rate of TAG oxidation. This, in
turn, leads to a reduction, in scrum TAG levels.

7.7. SYNTHESIS AND OXIDATION OF KETONE BODIES. KETONEMIA,

KETOACIDOSIS
Fatly acids released from adipose tissue serve as the major fuel for the body during
fasting. But some tissues cannot utilize kitty acids. The brain docs not utilize fatty acids
because of the limited transport of fatly acids across the blood-brain barrier. Although
the brain accounts for only 2% of the body weight in the adults, it consumes about
20% of the total energy in the resting body. This large energy demand is covered by
glucose under ordinary conditionsand by glucose and ketone bodies during prolonged
fasting. Ketone bodies arc synthesized mainly in. the liver and used as fuels during
fasting, starvation and long term exercise. As glucagon or epinephrine stimulate TAG
degradation in the adipose tissue, fatty acids level rises in the blood. The hepatic laity
acid utilization depends on substrate availability and therefore increases when laity
acid levels arc elevated in blood. As the rate of p-oxidation also depends on substrate
availability, it increases in the hepatocytes. About 50% of the fatty acids that enter the
liver arc convened to ketone bodies and only about 50% arc oxidized to CO. and H,O.
The initial substrate for ketone bodies synthesis is acetyl-CoA generated from
p-oxidation in the liver mitochondria (Fig. 7.21). The condition required for activation
of ketone bodies synthesis is the accumulation ofacetyl-CoA in the mitochondrial
matrix. When the rate of P-oxidation increases during fasting and starvation the
production of acetyl-CoA also increases. The subsequent oxidation of acetyl-Co A in
the TCA cycle lowers as the cvelc is inhibited by a high level of AT Pa nd high NADH/
NAD ratio produced by p-oxidation. That prevents the oxidation of acctvl-CoA in
the TCA cycle; also, the level of oxaloacctate required for entrance of acetyl-CoA
in the TCA cycle is low. due to its utilization in gluconeogenesis in the liver during
fasting. Il results in accumulation ofaceLyl-CoA in mitochondrial matrix and divert it
into ketone bodies synthesis.
Ketone bodies synthesis starts from condensation of two acetyl-CoA molecules
to form acetoacctyl-CoA (Fig. 7.22). The reaction is catalyzed by p-kctothiolasc. In
the next reaction, HMG-CoA synthase condenses acctoacetyl-CoA with another
molecule of acetyl-CoA to form p-hydroxy-p-mcthylglularyl-CoA (HMG-CoA).
H MG-CoA lyase then cleaves HMG-CoA to yield acctoacctalc and acetyl-CoA. Some
of acctoacctaie is reduced to p-hydroxy butyrate by p-hydroxybutyrate dehydrogenase.
The active oxidation of fatty acids generates NADH, part of which is used in the
reduction of acetoacctatc. Ketone bodies, acetoacctatc and P-hydroxy butyrate, arc
released to blood and transported to the other tissues. Concentration of ketone bodies
346 Chapter 7. Lipid metaD ol ism

Blood
Brain

Heart
Glucose
Muscles

Glucose
I
Acetyl-CoA Acetoa estate - jyhydroxyDutyratG

Oxalacetate
;
1
tca-
cyclo /
;
I
Acetoacstyl-CoA

Z Mitochondrion

Amino acids

Hepatocyte Cytosol

Fatty acids Adipocyte

Fig. 721. Fatty acids, ketone bodies aiid glucose metabolism in fasting

in blood increases and they become an important energy source for many tissues,
including the brain, thereby sparing the use of glucose. But ketone bodies cannot be
oxidized in the erythrocytes because they tack mitochondria. Cells transport both
acctoacciatc and p- hydroxy butyrate from the blood in the cytosol and then into the
mitochondrial matrix. There p-hydroxybutyrate is oxidized by p-hydroxybutyrate
dehydrogenase back to acctoacciatc (Fig. 7.23). This reaction produces NADH which,
can be oxidized in ETC. Acctoacctate is activated by accepting CoA from succinyl-
CoA. The reaction is catalyzed by succinyl-CoA: aceloacetatc-CoA transferase. The
enzyme is absent in the hepatocytes, therefore, the liver produces ketone bodies, but
cannot utilize them. Next reaction is cleavage of acctoacctyl-CoA into two acctyl-
CoA. which enter the TCA cycle and arc oxidized producing ATP. The energy yield
from oxidation of acctoacciatc, in theory, is equivalent to the yield for oxidation
of 2 molecules of acetyl-CoA in the TCA. cycle minus the energy for activation of
acctoacciatc (2* 12-1-23ATP). Oxidation of p-hydroxybutyrate produces one
additional NADH. Therefore, the energy yield from oxidation of p-hydroxybutyrate is
26 molecules of ATP but due to the dissipation of energy in reality it is approximately
21,5 molecules of ATP.
Regulation of ketone body metabolism. The rate-limiting enzyme of ketone bodies
synthesis is 3-hydroxy-3methylglutaryi-CoA synthase (HMG-CoA-synthase). The
synthesis of this enzyme is stimulated by fasting, insulin deficiency and excess dietary
fat consumption. Fatly acids levels arc elevated in blood in these conditionsand a large
amount of fatty acids arc taken up by the liver. Fatty acids arc powerful inducers of
HMG-CoA synthase. The inhibitor of H MG-CoA synthase is free CoASH. The level
7.7. Syntnesis artel Oxidation of Ketone Bodies. Ketonemia, Ketoacidosis 347

O O

CHg-C-SCoA + CH3-C-SCoA ( 2acefyl-CoA |

J^COASH

p-kstotnidase

o
I
CH-C

Cbk
1
C-O
SCoA O
K- CH3- C - SCdA
HMGJDoA
synthase
P* CoASH

S-HydfCwy-S-methyl
-glulafyl-CoA (HMG-CgA)

HMG-CoA
lyase
Acetyl-CoA

0
CH3 C-CHa- C -O’ (Acetoacgr^J

'/* NADH + H*
(Wiydraxybutyfata r _
Spontaneous
dehydrogenase
NAD*

CH3—

•' 0-^nydrOxy faityr aig ■ Acetone?

Fig. 7.22. Ketone Dodies synthesis. p-Hydroxybutyraie and acetoacetale are ketone Dodies wtiicfi
oxidation produces ATP; acetone is produced Dy nonenzymatic decarboxylation of acetoacetate in
case of nigh levels of ketone Dodies in blood

of CoASH in hepatocytes decreases as it is used for conversion of fatty acids entering

the cells to their acyl-Co A derivatives. Thus, the activity of HMG-CoA synthase
increases leading to the activation of ketone bodies synthesis. The normal range of
ketone bodies in plasma is 2-3 mg/dl (OJ -0,2 niM/1). During the prolonged fasting
it rises up to 50 mg/dl (2-3 mM/l) and in patients with diabetes mcllitus up to 500-
700 mg/dl (8-20 m M/1). Accumulation of ketone bodies in blood is called ketonemia,
secretion in urea — ketonuria. Kcloncniia develops as a result of disbalance between
overproduction of ketone bodies and capacity of their oxidation by peripheral tissues.
348 Chapter 7. Lipid metaD ol ism

Oh o
✓
Ch3—C— Cnr C — O"

i D-fl-hydroxy tiu:yraiej

NAO+
|54iydraxytHjlyrate
dehydrogenase
NADrt -t-H+
a o
E I
CH3— C—CHn—C— 0"
t Ace La ace Sate )

Succinyl-CoA: Sucdnyl-CoA^-
AcelcacelJale-CoA
transferase Sucdnale
Q 0
1 1
Crt3 — C“ CH^- C SCoA

(Acetoapelyl-CoA ]

CoASH
Thiolase

O O
I
Cl-h-C -SCoA + CH.3- C - SCdA
2 x C Acetyl-CoA ]

Fig. 723. Ketone bodies oxidation

Ketoacidosis. Ketone bodies arc water-soluble organic acids and dissociate

releasing protons:

OH OH
I _______ ~ I
ch3—c—ch?— cooh -*------- ► ch3—c—ch2— COO + H
|3 - hydroxybutyratc

Accumulation of ketone bodies in blood leads to ketoacidosis, h can be a life­

threatening symptom if blood ketone bodies concentration approaches 20m M.
Human body lias different mechanisms to excrete extra ketone bodies. Pan of
accloacclatc undergoes slow spontaneous nonenzymalic decarboxylation to acetone
(Fig. 7.22). Acetone is not oxidized in a body but can be excreted in urine or exhaled
by the lungs, thus, decreasing the risk of ketoacidosis. In case of ketoacidosis, acetone
gives a characteristic odo to the patient’s breath.. Kc-tonuria is the important pathway
for excretion of ketone odour and test lor ketone bodies presence in. the urine is used
for diagnosis of diabetes me Hit us.
7.7. Synthesis artel Oxidation of Ketone Bodies. Ketonemia, Ketoacidosis 349

Review test
1. TAG mobilization from the adipose tissue. Match the figure with the letter.

□
Praeii Kinase A
inactive
13
£3
Prosin Kinase A
aciiv2

Hormone sensitive liiase

QudBDOn
miissues

Adipocyte Blood

A. cAMP.
B. MAG.
C. Insulin.
D. Glucagon.
E. L.P lipase.
2. P-Oxidalion of fatty acids. Match the figure with the letter.
o

CHnwCHz- C'r+2- Ch- C-SCcA Acyi-CoA

Enoyl-CaA

©
Oh O
ni 1
CH3W0+2— CH—CH?— C - SCcA |l hydic^j*zy: Cn-'

naef

naDh+h*
©

fl-KciMcyfrCaA

CcASH fr-KcfcfnLrx

1
i!-SCnA + CHj —C-SCoA
CHj^—
I
TCA-cyoc
350 Chapter 7. Lipid metaD ol ism

A. p-hydroxy acyl-CoA dehydrogenase.

B. p-kctoihiolasc.
C. Acyl-CoA dehydrogenase.
D. Enoyl-CoA hydratase.
E. NADH dehydrogenase.
3. Ketone body metabolism. Match the figure with the letter.

GH2 —C-SCoA + CHj—C- SCoA [ 2 acelyi SCoA ]

fi-kelotrialasc
Is* CoASH

{Acetoace tyf-CoAj

K—CHs— C - SCoA

P* CoASH
OH 0
I
CHa —C— Crtj— C — O

CH?
C«"O
4
SCoA

ch3-c—ci-k—c—o-
? L—£-----J

OH F
1
MADH + H*

Ch3“ C — CH?- cT- O"

fb~ p-hydrorybutyrate'

A. Acctoaceiatc.
B. Acetone.
C. Fatty acvl-CoA.
D. H MG-CoA.
E. Acetate.
7.7. Synthesis artel Oxidation of Ketone Bodies. Ketonemia, Ketoacidosis 351

4. Ketone body metabolism. Match the figure with I he letter.

CHa—C—Crtj—

f 2 1
L—NAD+

p* NADH + H*

0 0
1 1
Ch3— C—CMj—C — O"

Sucdnyl-CoA

p* Sucanale
o Q
1 1
ch3-c-- CHjt’- G’-’ SCoA.

AceLoacetyt-CoA]

CaASH

A. Acetyl-CoA.
0
1
r
GH3—C - SCoA + CH3—C - SCoA
□
I

B. p-hydroxybutyraic.
C. Acctoacctatc.
D. HMG-CoA.
E. Fatty acyl-CoA.

Situational problems
1. Following a physician recommendation, an overweight man, 40 years old,
decided to start a healthy lifestyle: daily running for an hour, fat consumption
reduction Lip to 20 g in a day, as well as a reduced amount of *fast carbohydrates*.
In result, he lost 2.5 kg of his body weight during the following two weeks. What
metabolic pathway must be accelerated to cause such fat accumulation drop in
this man? Answer the questions and do the following tasks:
a) what is an adult norm for fat and carbohydrates daily consumption?
b) draw the scheme, showing the influence of persistent physical exercising on
lai metabolism in adipocytes;
c) draw the scheme of the metabolic pathway, where products of lipolysis arc
used in skeletal muscles during persistent physical exercise.
2. How docs the fatty acids concentration changes in the blood during prolonged
fasting? To answer the question:
a) name the hormone which manages metabolism in. this condition;
b) draw the scheme of this hormone action on the adipose tissue:
352 Chapter 7. Lipid metaD ol ism

c) draw the scheme of the main metabolic pathway providing energy Tor the liver
during prolonged lasting.
1 After 12 days of starvation, a man reduced his weight by 4 kg. The doctor
prescribed him biochemical blood test to analyze the level of lipids in serum
blood. The results showed an elevated level of free fatty acidsand triacylgtyccrols.
Explain the results of the biochemical blood test. For that:
a> name the main, hormone which manages metabolism during starvation;
b) draw the scheme of this hormone action on the adipose tissue;
c) name the possible pathways of fatty acids usage in the liver during starvation
and write down the proper schemes.
4. A sportsman is doing a 10 km marathon for 1.5 hours. What changes in lipid
metabolism in adipose tissue and muscles arc triggered by long-term physical
activity? To answer the question:
a} name the hormone, controlling the lipid metabolism in adipocytes and write
down the scheme of the process;
b) explain. how some of the products of lipid mobilization, transported from
adipocytes to the bloodstream, would be utilized by skeletal, muscles and liver
in these conditions;
c) calculate the ATP yield in the result of palmitoleic acid oxidation.
5. A long-distance runner trained for a. year to achieve better results. As he trained,
he increased his mitochondrial capacity, as well as his oxygen delivery to the
tissues. Why did these physiological mechanisms of adaptation to intensive
physical activity allow him to win the competition? For the answer
a) name the hormone which controls the metabolism during physical activity
and the fuel molecules secreted to blood from adipose tissue during prolonged
physical activity;
b) draw the scheme of their oxidation in the muscles;
c) explain why the improved oxygon delivery’ to the tissues will affect this
pathway.
6. The popular those days energy drinks have caffeine, theophylline, and
carnitine among other compounds. Caffe inc and its analog theophylline
inhibit the enzyme phosphodiesterase (Ch. 4). Such drinks arc made fizzy
(carbonated) to escalate its absorption and the effect. What arc the effects of
these components on a lipid metabolism? Answer the questions and do the
following tasks:
a) wrhal metabolic cascade in the adipose tissue would be influenced by caffeine
and theophylline? Explain the mechanism of their action:
b) draw the scheme of metabolic pathway for which carnitine is required:
c) explain the name ^energizer* for this sort of drinks.
7. An experimental animal was tested for fatty acids concentration difference on
1st and 30th minute of the workout: in the inflow blood, feeding the actively
working muscle, versus the outflow blood, coming from this muscle. In what
case difference will be more prominent? To answer
a) draw the reactions of the metabolic pathway, responsible for the difference in
the fatty acid concentrations in these conditions;
7.7. Synthesis ana Oxidation of Ketone Bodies. Ketonemia, Ketoacidosis 353

b) what arc die mechanisms of this pathway Lip- and down-regulation?

c) how and why tissue hypoxia would change that metabolic pathway rate?
8. Ketone bodies wore found in the urine of the patient with diabetes mellitus.
Explain the sequence of metabolic changes resulting in ketonuria. For that
answer the question and do the following tasks:
a) what arc the ranges of ketone bodies in blood of the healthy people and the
patients with diabetes mellitus?
b) explain the main changes in hormonal regulation of patient with diabetes
mellitus;
c) explain why the level of free fatty acids in the blood is elevated:
d) name the pathways of lipid' metabolism which becomes more active in patients
with diabetes me Hit us;
e) draw the scheme of ketone body sy nthesis and oxidation.
9. A man has been found unconscious on the street and delivered to hospital by
an emergency service. He was very slim and smelled acetone. Blood test results
showed the following: |glucosc| — 2.8 mmol/L, |ketone bodiesl — 40 mg/dL.
What arc the possible causes of such a condition? Answer the questions and do
the following tasks:
a) what is the role of ketone bodies in metabolism;
b) give an example of sit nations, when their synthesis is being elevated;
c) explain the origination of the substrates for the ketone bodies synthesis and
write down the schemes of their synthesis and oxidation:
d) explain the risk of ketone bodies accumulation in the blood:
e) what could be the possible reason here forgetting unconscious?
10. The patients with a genetically determined me di urn-chain acyl-CoA
dehydrogenase (MCAD) deficiency suffer from episodes of profound fatigue
associated with vomiting, which is happening if they fasted for more than
8 hours. They have hypoglycemia, elevated level of free fatty acids in the
blood but ketone bodies were below normal. Explain the development of these
symptoms. For that answer the question and do the following tasks:
a) why hypoglycemia occurs in the patients fasting for more than 8 hours?
b) why the level of free fatty acids is elevated in blood? Write the scheme
explaining this symptom;
cl draw the scheme of fatty acid: metabolism, which is blocked in case of this
genetic disorder.
I I. Four years old boy experiences problems in dealing with physical load. Muscle
biopsy has shown that carnitine concentration is 4 times less then, normal. Lipid
vacuoles have been found in the cytosol of the muscle cells. What arc the possible
causes of such a condition of the padent? For the answer:
a) draw the reactions of the specific metabolic pathway, disturbed in the patient;
b) describe the role of carnitine in this process, supporting the answer with an
appropriate scheme;
c) describe the molecular mechanisms underlying the process of fat accumulation
in the myocytes of the patients with this disease.
354 Chapter 7. Lipid metaD ol ism

7,8. STRUCTURE METABOLISM AND BIOLOGICAL EFFECTS

OF EICOSANOIDS
The eicosanoids, which include the prostaglandins (PG), thromboxanes (TX)
and leukotrienes (LT), arc potent regulators of cellular functions and arc produced
by almost every cell in a body. Eicosanoids have a short half-life in the range of few
minutes or less, before being inactivated and excreted. Therefore, they act as paracrine
(locally active) or autocrine (acting on the same cell from which it is released)
regulators through a family of transmembrane receptors. Eicosanoids regulate many
cell functions such as smooth muscle contractility, various immune and inflammatory
responses, blood coagulation, modulation of hormonal activity.
The structure and nomenclature of eicosanoids. The most common precursor of
the eicosanoids (^eicosa* is the Greek word for 20) is arachidonic acid-containing
20 carbons (20:4 A5,8,l IJ4 cu6). Besides the arachidonic acid, other 20 carbon fatty
acids (20: 5 and 20:3) arc also the precursors of the eicosanoids. Prostaglandins arc
the derivatives of these fatty acids containing five carbon (cyclopcntanc) ring In
addition to this ring, each of prostagland ins has hydroxyl group at carbon 15, a double
bond between carbons 13 and 14 and various substituents in the ring (Fig. 7.24).

Fig. 724. The structure of some eicosanoids derivatives of arachidonic acid

The nomenclature of prostaglandins is based on substituents to the cyclopcntanc

ring (e.g., the group is substituted with alpha, beta-unsaturated ketones, «E*
group is substituted with beta-hydroxyl ketone and with 1,3-di.ol). The subscript
I, 2 or 3 (c.g.. PGEj) indicates the number of double bonds in the side chains
7.8. Structure Metabolism and Eiologicai Effects of Eicosanoids 355

(Fig. 7.25). The Greek letter subscript in the F series (PGFa) denotes the position
of hydroxy] group below the plane of the ring at carbon. 9, as does hydroxyl group
at carbon I I. The types of prostaglandin, as designated by the capital letter, have
different and sometimes antagonistic biological effects. The number of double bonds
in the side chains affects the potency of the prostaglandin but not the kind of effect
on the particular target tissue.

C2D2£mi,14
(precursor)
(jj6

Eicosatrienoic acid
(dihomo - y - linolenic acid} PGE!

CO0H
CxrSASB, 11,14, E7
(precursor)
£o3

[Eicosapentaonoic acid)

Fig. 7.25. Prostaglandins of the 1.2 aim 3 series aim their precursors

Thromboxanes (TXT resemble prostaglandins in structure except that they contain

a six-membered ring that includes an oxygen atom. The most common thromboxane,
TXAr contains an additional oxygen atom attached to both carbon 9 and carbon II
of the ring. The thromboxanes arc synthesized in platelets and were named for their
action in producing blood clots ( thrombi).
Leukotrienes (LT) arc also synthesized from the same precursors but by another
enzyme — lipoxygenase. Leukotrienes arc named so because they arc synthesized
in leukocytes and contain three conjugated double bonds, which arc three double
bonds in series. One double bond is non-conjugated and the total number of double
bonds in a molecule is 4. The subscript 4 denotes four double bonds in the molecule
(LTAp. Other leukotrienes arc formed from LTA, by addition of another hydroxyl
group (LTl?4) or by the addition of glutathione and its fragment (LTC4 LTD4 LT E4).
The main biological functions of leukotrienes arc increasing of vascular permeability,
bronchoconstriction, leucocyte aggregation, stimulation of interferon and interleukin
secretion.
The functions of the different types of eicosanoids arc listed in Table 7.4.
356 Chapter 7. Lipid metaD ol ism

Tatle 7.4. The biological erieds of me main eicosanoids {derivatives of arachidonic acid)

1 Bctmw*1 j Major sfe(s) nt syrtfaesk

PGDS Mast cells Vasodilatation

PGEg Stomach, kidney, heart, endometrium, Suppress gastric acid secretion, reduce the risk
vascular endoth elial cells of gastric and duodenal ulcer
White blood cells Vasodilatation, induction of uterine contractions,
inhibition ol platelet aggregation
Mediator ol inflammation

PGFga Kidney, spleen, heart, endometrium Vasoconstriction, smooth muscle contraction,

Uterine contraction

PGHg All types of cells Precursor to TXA? and Ey induction of platelet

aggregation and vasoconstriction

PG!, Heart, vascular endothelial cells Inhibits platelet aggregation, induces

vasodilatation

TXA, Platelets, white blood cells Induces platelet aggregation and vasoconstriction
Mediator ol inflammation

TXfia Platelets Induces vasoconstriction

ltb4 Monocytes. basophils, neutrophils, Induces leukocyte chemotaxis and aggregation;

mast cells eosinophils, epithelial cells increase vascular permeability

Monocytes and alveolar macrophages, Increase vascular permeability;

basophils, neutrophils, mast cells bronchoconstriction. Predominant component of
eosinophils, epithelial cells stow-reacting substance of anaphylaxis

Monocytes and alveolar macrophages., Induces vasodilatation and bronchoconstriction.

neutrophils, mast cells eosinophils, Predominant component of slow-reacting
epithelial cells substance of anaphylaxis

Mast cells and basophils Induces vasodilatation and bronchoconstriction.

Predominant component of slow-reacting
substance of anaphylaxis

PGDj— prostaglandin D,, PGJ_. — prostacyclin lr TXA, — Eftrombaxa^ie A ? LTB f—laufcotriene Br

The synthesis of the eicosanoids. Arachidonic acid, the main precursor of the
eicosanoids, yields the 2 series (2 double bonds in rhe side chains) of prostaglandins
(Fig. 7.25). The I. and 3 series of prostaglandins arc derived from cicosairienoic
acid — C 20:3 I AS, 11.14 cub) and cicosapentaenoie acid — C 20:5 (A 5,8,11,14,
17 m3). These acids arc cstc rifled to phospholipids located in the lipid bi layer of
the plasma membranes of the cell. The synthesis of the eicosanoids starts with the
rate-determining hydrolyzis of arachidonate (or another 20-carbon polyunsaturated
fatly acid) from the 2nd carbon of glycerol of membrane glyccrophospholipids. The
reaction is catalyzed by a receptor-activaicd phospholipase A3 (PLA2) (Fig. 7.26).
Molecules that act as primary signals for eicosanoid synthesis bind to the cell
membrane receptors and activate the phospholipase Ar Some primary messengers
activate inositol triphosphate system of signal transduction in the target cells.
The formed second messenger diacyiglycerol (DAG) activates membrane-bound
phospholipase A, which hydrolyzes phosphatidylcholine located in the membrane.
Released arachidonic acid or another 20-carbon polyunsaturated fatty acid arc the.
7.8. Structure Metabolism and Eiologicai Effects of Eicosanoids 357

substrates for an enzyme cyclooxygenase. Cyclooxygenases known as COXs arc

bi function al enzymes that con La in both cyclooxygenase and peroxidase activity. At
first, arachidonate undergoes the reaction of cyclization by the addition of two oxygen
molecules. The product of the reactions hydroperoxide cyclic endoperoxide (PGC,),
is the substrate for the hydro peroxidase, producing the hydroxy cyclic endoperoxide
(PGH,). PGH2 is the substrate for subsequent enzymatic modifications leading to
the formation of prostaglandins PGD,, PGE2, PGE.a, prostacyclin (PG13) and
thromboxane (TXA.).
Cyclooxygenascs exist as distinct iso forms referred to as COX-1 and COX-2. COX-
1, expressed constitutively in most cells, is the dominant source of prostaglandins
and thromboxanes for « normal* physiologic functions (Table 7.4). COX-2, induced
by proinflammatory stimuli, a variety of cytokines and growth factors, is the more
important source of eicosanoid synthesis in inflammation and proliferative diseases,
such as cancer. However both enzymes contribute to the generation ofautoregulatory
and homeostatic eicosanoids, and both can contribute to eicosanoids rclcazc, during
inflammation.
Cyclooxygenase pathway produces prostaglandins (PGEr PGFr PGA^),
prostacyclin (PG12), and thromboxane (TXA,). Lipoxygenase pathway produces
leukotrienes (LTA4).
Arachidonic acid is converted to different types of eicosanoids by a variety of
enzymes with activities that vary among the tissues. This variation explains why
different types of the cells, such as those in the vascular endothelium, synthesize
prostaglandins 1. and EU PG I, and PGE,), whereas platelets synthesize thromboxane
A, (TXA2) from the same precursor.
Another lipoxygenase pathway produces leukotrienes, lipox ins and
hydroperoxy eicosa tctracnioc acids (H P'ETEs) from the same precursors. The
lipoxygenase catalyzes the incorporation ofan oxygen molecule onto a carbon ofone of
the several double bonds of arachidonic acid, forming a hydroperoxy group (-OOH).
If this group is added to the 5lh carbon, 5 — HPETE (hydroperoxyeicosalctrocnoic
acid) is formed. Then the unstable hydroperoxy group is convened to hydroxy
group and three double bonds become conjugated forming LTA4 leukotriene A4
(Fig. 7.26). Main biological functions of leukotrienes arc: increasing of vascular
permeability, bronchoconstriction, leucocyte aggregation, stimulation of interferon,
and interleukin secretion. Leukotrienes C4, l)4, and E4 which were originally
characterized as the slow-reacting substance of anaphylaxis, are responsible for
the bronchoconst riel ion in asthma. The main symptoms of bronchial asthma arc
the result of inflammation involving the mucosal and smooth muscle layers of the
respiratory tract. Hypersecretion of leukotrienes and prostaglandins causes the main
symptoms of bronchial asthma.
The third pathway, catalyzed by the cytochrome P45(L is responsible for the
synthesis of epoxides and HETEs. The biological activities of these compounds
include action on endocrine, vascular, and renal systems.
358 Chapter 7. Lipid metaD ol ism

0 CHL—O^C— r,
,-Uo-Ah
Ahj— O—®—Chriine

Membrane phospholipid
Phospholipase A2
Q Glucocorticoids
cyt
Epoxides -P4b0

Lipoxygenase
Arachidonic acid
(Cxr-tAS.fiJ1,14;)

Cyclooxygenase
—I Aspirin and other NSAJDs

Prostaglandins

Fig. 7.26. Synthesis of Ute eicosanoids from arachidonic acid

The eicosanoids have a wide variety of physiologic effects ( Fable 7.4) which arc
initiated though the interaction of the eicosanoid with a specific receptor on the
plasma membrane of a target cell. These receptors arc connected with adenylate
cyclase or guanylatc cyclase systems or cause an increase in the level of calcium in
the cytosol of the target cells acting through inositol triphosphate system. Some
eicosanoids modulate the degree of systems act ivatio n in response to other stimuli. If
the eicosanoid binds to the stimulatory subunit connected with the receptor the effect
of the stimulus is amp I i heated. If it activates inhibitory subunit, the cellular response
is reduced. Thus, the eicosanoids contribute to the regulation of the cellular functions
by hormones and other primary messengers.
Many eicosanoids have antagonistic biological activities. Prostacyclin tPGlj
and thromboxane (TXAp arc potent antagonist regulators of platelet aggregation,
thrombus formation and smooth muscle tone. Normal vascular endothelium
7.8. Structure Metabolism and Eiologicai Effects of Eicosanoids 359

produces prostacyclin I, which is synthesized from PGHJn a reaction catalyzed by

PG I synthase. PG I .causes vasodilation of blood vessels smooth muscles and inhibits
platelets aggregation. As a result, blood doesn’t coagulate (Fig. 7.27). Thromboxane A2
is synthesized in platelets also from PG H, but in the reaction catalyzed by thromboxane
synthase. TXA.arc produced by platelets after their activation through the contact
with the injured endothelium. The injury of endothelium usually is a result of the
formation of atherosclerotic plague which destructs the endothelial cells. The released
TXA., is potent vasoconstrictor and a stimulator of platelet aggregation (Fig. 7.28).
As a result, thrombus formation is rapidly accelerated at site of vascular injury and
the walls of arteries contract. Such thrombus may cause sudden total occlusion of
the vascular lumen, causing acute ischemic damage to tissues. Thus, the myocardial
infarction can develop if the atherosclerotic plague is located in artery of the heart.

Fig. 721. Regulation of vascular smooth muscle tonus Dy prostacyclin ir intact vascular endothelial
cells produce PGi?which inhibits platelet aggregation and causes vasodilation. Nitric oxide released by
me enuotneiiai ceils nas the same effect on tonus of smooth muscle ceils

Fig. 728. Releasing of thromboxane TXA,. and activation of platelet aggregation

360 Chapter 7. Lipid metaD ol ism

Daily consumptions of low-dose aspirin inhibit the cyclooxygenase and block the
production of thromboxane TXA2 thus decreasing the risk of thrombus formation and
preventing the acute myocardial infarction (Fig. 7.29). A lot of researches report about
the effect of the diet that include cold-water fish with a high content of polyunsaturated
fatty acids ofco-3 family (C:20 A 5,8 J 1,14,17) on prevention of heart attack. This diet
increases the content of to-3 fatty acids in membrane phospholipids. In this case, the
cicosapentacnoic acid becomes the initial substrate for thromboxane synthesis and
more TXA, is produced relative to TXA,. TXA.S is less effective in stimulating platelet
aggregation than its counterpart TXA, But the discussion about benefit oftu-3 fatty
acids is still continued.
As the prostaglandins arc important in mediating the inflammatory response, drugs
that block prostaglandin synthesis should provide relief from inflammation symptoms.
The cyclooxygenase enzyme is inhibited by all nonsteroidal anti-inflammatory drugs
(NSAIDs) such as aspirin. Acetyl group from aspirin covalently binds to the enzyme,
irreversibly inactivating it.

Fi^ 7.29. Inactivation of cyclooxygenase hy aspirin. Acetyl group of aspirin binds by covalent hond to
Hydroxyl in the active site of cyclooxygenase causing its irreversible inhiDition

Other NSAIDs act as reversible inhibitors of cyclooxygenase. NSAIDs blocks

the activity of both cyclooxygenase isoenzymes, COX-1 and COX-2. The inhibition
of COX-1 causes the gastrointestinal side effect (stomach ulcers) through the lower
synthesis of gastro protective prostaglandins and anti plate let side effect commonly
associated with NSAIDs use. To avoid these effects some drugs inhibiting only COX-
2 were synthesized. These drugs act as potent anti-inflammatory agents without the
side effect of aspirin and other NSAI Ds. More potent anti-inflammatory agents arc
glucocorticoids which include the hormones cortisol, cortisone and their synthetic
7.8. Structure Metabolism and Eiologicai Effects of Eicosanoids 361

analogs such as dexamethasone. These steroids reduce inflammation by inhibiting the

action of phospholipase Ap which is responsible for the release of arachidonic acid
for prostaglandin synthesis. This action is mediated through the protein annexin-1
(or lipocortin). Glucocorticoids such as dexamethasone also inhibit expression of
cyclooxygenase 2, which is induced in inflammatory cells. Glucocorticoids inhibit the
recruitment of while blood cells into affected aria and limit the ability of these cells to
release the eicosanoids, which enhance the inflammatory process.

Review test
I. Match the figure with the letter:

A. TxA,.
B. TxA^.
C PGEr
D. PgCr
E. arachidonic acid.
2. Match the figure with the letter. Inhibitors of the enzymes:

Membrane
pnospnoiipids
L*—Phospholipase

Aracnidonic acid

Leucotriens Prostaglandins
362 Chapter 7. Lipid metaD ol ism

A. Glucocorticoids.
B. Aspirin.
C. Vitamin D.
D. Vitamin E:-E glucagon.
3. Choose lhe correct answer. The Ley enzyme of proslagtan din synthesis is:
A. Lipoxygenase.
B. Cyclooxygenase.
C. Peroxidase.
D. Phospholipase Ar
E. Adenylate cyclase.
4. Choose lhe correct answer. The key enzyme of leukotriene synthesis is:
A. Lipoxygenase.
B. Cyclooxygenase.
C. Catalase.
D. Phosphodiesterase.
E. Adenylate cyclase.

Situational problems
1. Rare side effect of aspirin is the development of acute asthmatic bronchospasm
after taking a pill ofaspirin. Why can this coni plication can develop? For that do
the following tasks and answer the question:
a) explain the mechanism ofaspirin action;
b) draw the scheme needed for the explanation ofaspirin action on the metabolic
pathway;
c) explain using the scheme the possible cause of this side effect of aspirin;
d) why the administration of glucocorticoid analog dexamethasone significantly
reduced the symptoms?
2. Aspirin ceases the symptoms of inflammation and reduces the rate of blood
coagulation. How the daily consumption of low-dose aspirin (cardio aspirin.)
can prevent acute myocardial infarction?
For the answer:
a) name the enzyme inhibited by aspirin;
b) explain the mechanism ofaspirin action on the enzyme;
c) name the eicosanoids which synthesis is inhibited by aspirin:
d) explain why the rate of blood coagulation is reduced despite lhe aspirin
inhibiting simultaneous synthesis of the both, eicosanoids act ingas antagonists
in blood coagulation.

7.9. GLYCEROPHOSPHOLIPIDS AND SPHINGOLIPIDS, BIOLOGICAL

FUNCTIONSAND METABOLISM
Glycerophospholipids and sphingolipids arc amphipathic molecules containing
both polar and nonpolar regions. Glycerophospholipids contain glycerol as a backbone
and phosphate group: sphingolipids contain amino alcohol sphingosine as a backbone
(Table 7.1) and only one of them — sphingomyelin — contains phosphate group. Both
classes of the lipids arc the main structural compounds of the cell membranes, and
7.9. Giyceropnospnoiipias and Spnmgoiipids. Biorogicai Functions ano Metabolism 363

both playa role in the generation of lipid-signaling molecules. Glyccro phospholip ids
also form the phospholipid monolayer on the surface of blood lipoproteins.
Glyeerophospholipids (phosphoacylglycerols) arc the derivatives of phosphal id ic
acid (PA) containing glycerol two fatty acids and a phosphate group on the Ehird
carbon (Fig. 730). The initial steps in the synthesis ofglycerophosphol ip ids arc similar
to those of triacylglyccrol synthesis (Ch. 7.13). Glycerol 3 — phosphate derived from
di hydroxy acetone phosphate the product of glycolysis subsequently interacts with two
acyl-CoA to form phosphatidic acid. Then different polar «hcad» groups are added to
the phosphate.

"dl

zCH3‘

— Ch2—CHg—hf Ch3 iPnospnaiidydcrwiifei

”CH3-

Pnospnatidyt-
— CK-2—ch2—nh-3+
ethaoDlamine
- r Jffianoiamine J

Pncspnatkiyiinositoi
I 4^4>ispnospoate

■ 4. > :-:5l ri2~

Fig. 7.30. Phosphatioic acid as the precursor of glycerophosphnlipifls. FhosphatidiC acid is

aiacyigiycerolpnospnate; Fl! and R? are ine nydropnobic radicals of fatty acids; R? is usually
polyunsaturated. The polar heads- of giyceropnospnolipids include phosphate residue with the
attached hydrophilic group like serine, choline, etnanoiamine, inositol 4.5-bisphaspnate

Phosphatidylcholine t lecithin) is the main phospholipid of all cell membranes.

In most tissues the glycerophospholipids including phosphatidylcholine contain
poly u nsa t u rated fa it y acids, e ha t dele rm i nos i he flu id i ty of t lie i n nc r hyd rophob ic layer of
the membranes. This allows the transmembrane proteins to change their conformation
easily during functioning. But. phosphatidylcholine of the lungs alveoli surfactant has
unique fatly acids composition. Both fatty acids in. this molecule are saturated pal mi tic
acids (dipalmitoylphosphalidylcholinc). Dipalmitoyl phosphatidylcholine is the major
compound of lung surfactant. The surfactant is secreted by alveolar cells and reduces
the surface tension of the fluid film that lines the alveolar walls. As the radicals of
saturated fatty acids are not oxidized by reactive oxygen species (ROS) (Ch. 5) the
structure and properties of surfactant to remain stable despite the high oxygen levels
in the alveoli of lungs.
364 Chapter 7. Lipid metaD ol ism

[f the production of the surfactant is insuflicicm as it happens in preterm infants,

the alveoli collapse and breathing become difficult. As a result, respiratory distress
syndrome can develop, a condition that is responsible for 1.5-20% of neonatal
deaths. The maturity of a fetus’s lungs can be determined by measuring the lecithin/
sphingomyelin ratio in. amniotic fluid. The concentration of phosphatidylcholine
relative to sphingomyelin rises at 35 weeks of gestation, indicating pulmonary maturity.
The surfactant can be administrated to treat infants with respiratory distress syndrome
by an inhaler.
Phosphatidylinositol i PI) is an unusual phospholipid in that it often contains stearic
acid on carbon I and arachidonic acid on carbon 2. PL therefore, serves as a source of
arachidonic or 20:5 and 20:3 fatty acids providing the substrates for eicosanoid synthesis
when required. Phosphorylation of mem brane-bound phosphatidylinositol produces
phosphatidylinositol 4, 5-bisphosphatc (PIP j involved in signal transmission across
membranes.
Cardiolipin is formed from two molecules of phosphatidic acid csterified through
their phosphate groups Lo an additional molecule of glycerol. Cardiolipin is virtually
exclusive to the inner mitochondria] membrane, where it appears to be required for
the maintenance of certain respiratory complexes of the electron transport chain.
Platelet-activating, factor (PAD is an unusual ether glycerophospholipid which,
belongs to plasmalogcns. PAF contains a saturated alkyl group in an ether link to
carbon I and an acetyl group al carbon 2 of the glycerol moiety ( Tig. 7.31). With these
exceptions the other part of the structure is similar to the phosphatidylcholine. PAT
is synthesized within peroxisomes of phagocytic blood cells and is released from these
cells in response to various stimuli.. PAF binds to surface receptors and causes platelet
aggregation, hypotension and edema. PAF and other types of plasmalogcns arc also
involved in allergic reactions and inflammatory processes. Plasmalogen synthesis is
compromised in individuals with a genetic defect in peroxisome formation {Zellweger
syndrome k If defect of plasmalogen synthesis is severe enough, this syndrome leads
to death al an early age.

Fi^ 7.31 Structure of me prate let-activating factor

Degradation of glycerophosphoiipids is catalyzed by several phospholipases

(Fig. 7.32). One of them, phospholipase removes the fatty acid on carbon 2
of glycerol. This fatty acid is usually arachidonic acid, which is removed from
7.9. Giyceropnospnaiipias and Spnmgoiipids. Biorogicai Functions ano MetaDoiism 365

the membrane phospholipids in response to signals for the eicosanoid synthesis.

The released arachidonic acid serves as the precursor for eicosanoid synthesis.
Phospholipase A2 is also involved in the major repair mechanism for membrane lipids
damage by lipid peroxidation reactions (Ch. 5). As the arachidonic acid at the second
carbon of glycerol is polyunsaturated fatty acid, it can be oxidized by oxygen free
radicals. Phospholipase A, recognizes the partially oxidized fatty acid and removes it.
Acyl transferases then bind back a new arachidonic acid.

Priosproiipase D

Fig. 7.32. Bo nets cleaved by phospho lipases

Phospholipase C stimulated by binding of hormones and neurotransmitters to

the receptors of cell membrane, hydrolyzes phosphatidylinositol bisphosphatc {Pl Pj
to produce the second messengers DAG and inositol triphosphate (IPS) (Ch. 4).
IP3 mediates the mobilization of intracellular calcium and the activation of protein
kinase C, respectively, which act synergistically to evoke specific cellular responses.
These examples demonstrate that the degradation of glycerophospholipids by
phospholipases is required for other important functions of the tissues.
Sphingolipids. The first reaction of sphingolipid synthesis begins from the
serine and palmitoyl-Co A condensation with pyridoxal phosphate as coenzyme
(PLP). The product of the reaction is reduced with, the formation of amino alcohol
dihydrosphingosi.no (Fig. 7.33). Dihydrosphingosinc undergoes the dehydrogenation
by FAD-dope nd ent dehydrogenase producing sphingosine which gives name to all
groups of sphingolipids. Then long-chain fatty acid binds to the amino group of
sphingosine and finally ceramide forms. Ceramide reacts with phosphatidylcholine to
form sphingomyelin. Sphingomyelin is the main, compound of myelin sheaths around
the axons emanating from the neurons.
Glycolipids arc also sphingolipids as they arc derivatives of ceramide to which
various carbohydrate groups arc attached. Glycolipids arc located in the plasma
cell membrane with the carbohydrate groups extruded from the cell surface. These
carbohydrate groups serve as cell recognition factors and some of them act as the
antigens. The main functions of glycosphingolipids arc:
* intercellular communication;
» interaction of the cells and intercellular matrix;
366 Chapter 7. Lipid metabolism

► antigenic determination of the ABO blood groups;

► maintenance of the membrane structure.

PalmitoykCoA + serine'

CoA.SH
CO2
H-NADPH + 2H+

P NADP*"
Dinydrospnigosine
Acyl-CoA

t
[Ceramide]
COASH

I - Pnospnatidytchoiine

p DAG
Ceramide Choline

t------------------- -------------------- *1
O CH,

P—o—CH2—CHg— N4_ CH3

O" ch3

c—c

(CH2)n

ch3_____
Q~Spnin-goirye in

Fig. 7.33. Synthesis of sphingomyelin. Ceramide is spningosine with a fatty acid attached to its amino
group Dy an amide linkage (blue Dox). Sphingomyelin contains pnospnocnoiine the polar «nead»
of sphingomyelin

Synthesis of glycosphingolipids also starts from ceramide. Ceramide reacts with

active forms of sugars such as UDP-glucose or UDP-galactose to form cerebrosides
(Fig. 7.34). These reactions are catalyzed by specific giycosyltransfcrases. Cerebrosides
contain a single sugar, monosaccharide, attached to hydroxymethyl group of ceramide.
Cerebrosides can react with active form of sulfate to form sulfatidcs, the major form of
sulfolipids of the brain.
7.9. Giyceropnospnoiipms and Spnmgoiipids. Biorogicai Functions ano MetaDoiism 3G7

Fig. 7.34. Synthesis of glycosphingol ipids from ceramide. Glc — glucose: Gal — galactose; Gal MAC —
M-aceiyigaiactosamine; WAN a — N-acetylneuraminic acid; UDP — sugars, active forms of sugars;
CMP-NANA — active form of N-acetylneuraminic acid

Gangliosides contain oligosaccharides and N-acctylglucosaminc and

N-acetylgalactosamine. These glycolipids form the blood group determinants.
The most widely studied is the ABO blood group system. The surface of human
erythrocytes is covered by complex specific antigenic determinants which arc
glycolipids (or glycoproteins) with different structure of oligosaccharide moiety.
Genetic variation is a result of specific glycosyl transferases responsible for synthesis
of the hole rosaccha ride determinants. The H gene codes for fucosyIIransferase
which links fucose to a peripheral galactose at the nonreducing end of blood group
determinants (Fig. 7.35). Most individuals can. synthesize fucosyltransferase and
fucose is present in the structure of all ABO determinants. The A allele encodes an
N-acetylgalactosamine glycosyl transferase that adds N -acetylgalactosamine to the
galactose residue of the H substance. The B allele encodes a galactosyl transferase that
adds galactose to the galactose residue of the II substance. The 0 allele encodes an
active protein and type 0 individuals cannot attach either N-acetylgalactosamine or
galactose to H substance of blood group determinants. Thus, individuals of blood type
0 have only the H substance on the surface of the erythrocytes. Type AB individuals
have both alleles and produce both transferases. Thus, some of the oligosaccharide
determinates of their blood group substances contain N-acetylgalactosamine and
some galactose.
368 Chapter 7. Lipid metaD ol ism

Type 0

Ry. 7.35. Structure of blood group determinants. These structures are the same except mat type A has
N-acetyigaiactosamine (GatNAc), type B has galactose (Gai) and type 0 has neither. Fuc — fucose;
CicNAc — W-acetyiglucosamine

The knowledge about oligosaccharide determinates of blood group substances is

essential io blood transfusion practices.
AH sphingolipids arc degraded by lysosomal enzymes. Deficiencies of these
enzymes result in a group of lysosomal storage diseases known as the sphmgolipidosis
(Table 7.5).
7.9. Giyceropnospnaiipibs and Spnmgoiipids. Biorogicai Functions ano MeiaDolism 369

Table 7J5. Examples of sphingoliptaosis - the Inherited diseases connected wim deficiency of enzymes
involved in catabolism of sphingolipids

Tay-Sachs fi-Hexosaminidase Cer-Gk:-GaH NeuAc)-GalNAc Mental retardation, blind­

disease A (GM2 Ganglioside) ness, muscular weakness

Fabry's disease a-Galactosidase Cer-Gk-Gal-Gal Skin rash, kidney failure

(Globotriaosylceramide) (full symptoms only in
(Globosides) males: X-linked recessive)

Metachromatic Arylsulfatase A Cer-GaPOSpj 3- (SutfogaJactos Mental retardation and

feukodystrophy ylceramide) (Sulfatide) psychologic disturbances
in adults; demyelination

Krabbe's disease p-Galactosidase Cer-Gal (Galactosylceramide) Mental retardation; myelin

almost absent

Gaucher's disease fi-Glucosidase Cer-Gfc (Glu cosyice ramide) Most common lysosomal
(glucocerebrosi- storage disease.
dase) Enlarged liver and spleen,
erosion of long bones,
mental retardation

Niemann-Pick Sphingomyelinase Cer-P-choline Enlarged liver and spleen,

disease (Sphingomyelin) mental retardation; fatal in
early life

Fartefs disease Ceram idase Acyl-Sphingosine (Ceramide) Hoarseness, dermatitis,

skeletal deformation,
mental retardation; fatal in
early life

] n. case of the enzyme deficiency, its substrate is accumulated in the cells and finally
destroys it. As most of sphingolipids a re in the nervous tissue, the mental retardation is
specific for many types of sph ingo lipidosis.

Situational problem
L Phosphatidylcholine molecules arc the main lipid compounds of all cell
membranes. Phosphatidylcholine, is also an important compound of the lungs
alveoli surfactant hut its fatty acids composition differs from phosphatidylcholine
present in the cell membranes. Explain the difference in fatly acid composition
of these two types of phosphatidylcholine molecules. For the answer:
a) draw the structural formula of phosphatidylcholine of the lungs surfactant
and give the full name of this structure:
b) explain the functions of phosphatidylcholine in. the membranes of the cells
and in the lungs surfactant;
c) compare the fatty acids composition in both types of phosphatidylcholine and
explain how it correlates with their functions;
d) explain the mechanism of respiratory distress syndrome development in
premature infants;
c) indicate the possible way of treatment of these infants.
370 Chapter 7. Lipid metaD ol ism

7JO. CHOLESTEROL FUNCTIONSAND METABOLISM. REGULATION

OF CHOLESTEROL SYNTHESIS
Cholesterol metabolism is particularly important as its disorders result in the rnosL
spread disease in humans — atherosclerotic vascular disease. Cholesterol contains a
four-ring structure called the steroid nucleus ( Fig. 7.36), eight carbons aliphatic chain
at 17^ carbon of the ring D, and two methyl groups. The total number of carbons in
cholesterol is 27. Hydroxyl group at carbon 3 in the A ring allows to form cholesterol
ester by interaction with fatty acyl-CoA. The fatty acyl attached to hydroxyl group of
the cholesterol by ester bond is usually polyunsaturated. mainly linoleic. Cholesterol is
the only steroid precursor in human cells for the synthesis ofall steroid hormones, bile,
salts and active form of vitamin I), cholccalcifcrol. Cholesterol itself is a stabilizing
component of cell membranes with its hydroxyl group faced to water and Ibur-ring
hydrophobic structure immerse into the lipid bilayer of the membrane. Cholesterol
is most abundant in the tissues, especially the nervous tissue, that contains various
membrane lipids. Free (or uncstcriftcd) cholesterol is also the compound of the shell
of any lipoprotein transporting lipids by the blood.

Fql 7.36. Structure of cholesterol and cholesterol ester. A — formula and model of cnotestefoi. B —
formula of cholesterol ester. R is usually the radical of linoleic acid

Cholesterol is obtained from the diet or synthesized in most cells of the body buL
mainly in the liver (:= 70%) and intestine (Fig. 7.37). Smaller amounts of cholesterol
arc synthesized in the adrenal cortex and gonads being the precursor of steroid
hormones.
7.10. Cholesterol Functions and Metabolism. Regulation of Cholesterol Synthesis 371

________ _________
Usage and excretion Ceils

Synthesis of Excretion with Steroid hormone

vitamin D skin fat synthesis
of -10mg/24h -0Jg£24h -40 mg.''24h
0,S-0,7g/24h ____________

Fig. 7.37. Formation and distribution of cholesterol pool in a body

Absorption of dietary cholesterol. Most people in modern societies get close io 0.5-
1.0 g of cholesterol per day with food. Cholesterol is present only in the food of animal
origin. Plants contain phytosterols instead, like ergosterol in fungi and P-sitostcrol
in higher plants. These steroids are poorly absorbed from dietary sources and not
involved in mammalians’ metabolism. A vegan diet is essentially cholesterol free. Part
of food cholesterol is present as cholesterol esters. Cholesterol and cholesterol esters
being water-insoluble molecules arc emulsified by bile salts. Cholesterol esters arc
digested by the enzyme cholesterol esterase. This enzyme secreted by the pancreas
cleaves cholesterol ester to free cholesterol and fatty acid. These products together
with the products of TAG digestion are absorbed as mixed micelles in the intestine.
Approximately only half of dietary cholesterol entering the gut per day is absorbed.
Cholesterol absorption by intestinal cells is an important regulatory point in human
cholesterol metabolism. Although dietary steraoids arc absorbed by diffusion from
the intestinal lumen to enterocytcs, there is a special mechanism to remove excess
cholesterol and plant sterol from the enterocytcs. The transport of sterols out of
the enteiocyies occurs by the adenosine triphosphate (ATP) — binding cassette
proteins (ABC). These proteins use ATP energy for the transport of unwanted sterols
from the enterocytcs back into the gut lumen and cholesterol and other sterols are
eliminated from the body mainly in the feces. Patients with rare autosomal recessive
disease phytosterolemia have a defect in the function of ABC proteins and high
372 Chapter 7. Lipid metaD ol ism

levels of cholesterol and phytosterol in the blood. This accounts for atherosclerotic
cardiovascular disease in the patients. Some drugs that block cholesterol absorption by
intestinal cells arc used for the treatment of hypercholesterolemia.
In the intestinal epithelial cell, most of the absorbed cholesterol is converted again,
to cholesterol esters by enzyme ACAT (acyl-CoA — cholesterol acyltransferasc). This
enzyme catalyzes the transferor a fatty acyl from CoA to the hydroxyl group on carbon
3 of cholesterol (Fig. 7.36). Then free cholesterol and cholesterol esters together with
triacylglyccrols and other absorbed dietary lipids arc packaged into chylomicrons.
Most of TAG in chylomicrons is removed by LP-lipase in the blood capillaries of
peripheral tissues and cholesterol-rich remnant particles thus formed arc taken up
by the liver. The compounds of chylomicron remnants arc hydrolyzed by lysosomal
enzymes and cholesterol becomes available for usage by hepatocytes. Thus, most of
the dietary TAG goes to extra hepatic tissues but most of the dietary cholesterol is
delivered to the liver.
Cholesterol synthesis. Endogenous cholesterol synthesis amounts up to 500-
1000 mg/24h, depending on the dietary supply. The complex cyclic structure
of cholesterol is synthesized from simple initial precursor — acetyl-CoA. Other
precursors required for cholesterol synthesis arc NADPH as donor of hydrogen for
reduction reactions and ATP as an energy source. The precursors can be obtained
from the glucose catabolic pathways: acetyl-CoA from aerobic glycolysis and NADPH
from pentose phosphate pathway. Other sources of acctyl-CoA arc ^-oxidation of fatly
acids in ease of high content of fats in the diet and the oxidation of kctogenic amino
acids such as leucine and iso I cue inc.
The pathway of cholesterol synthesis is one of the longest in. human cells
and involves at least 30 enzymes. The synthesis of I mole of cholesterol requires
IS moles of acctyl-CoA, 36 moles of ATP and 16 moles of NADPH. All reactions
of cholesterol synthesis occur within the cytoplasm, although some of them occur
in the endoplasmic reticulum. The whole pathway is subdivided into several stages
(Fig. 7.38).
The first is the synthesis of mevalonic acid (mevalonate) from acetyl-CoA. In
this pathway two molecules of acetyl-CoA condense, forming acetoacetyl-CoA,
which then condenses with the third molecule of acetyl-CoA to yield the 6-carbon
molecule p-hydroxy-p-nicthylglutaryl-CoA (HMG-CoA). These reactions, catalyzed
by acctoaceiyl-CoA thiolase and HMG-CoA synthase- arc the same as the ones in
the synthesis of ketone bodies, although the latter process occurs only within the
mitochondria. In the next reaction, catalyzed by HMG-CoA reductase. HMG-CoA
is reduced to mevalonate. Hydrogen atoms required for this reducing reaction are
donated by two molecules of NADPH. H MG -CoA reductase is the key enzyme of the
pathway of cholesterol synthesis.
The second stage is the conversion of mevalonate into two activated isoprenes
A3-isopentenyl pyrophosphate and dimethylallyl pyrophosphate. Mevalonic acid
(mevalonate) al first is phosphorylated in reactions requiring kinases and ATP.
Subsequent decarboxylation leads to the formation of isopcntcnyl pyrophosphate and
di methyl allyl pyrophosphate which, arc the isomeric 5-carbon isoprene units. Three
ATP molecules arc required for this sequence of reactions.
7.10. Cholesterol Functions and Metabolism. Regulation of Cholesterol Synthesis 373

c>

CHa—€ SCcA
[Acetyf-CoAj
C- CHa— C—SCoA

[’■* CoASH
O
1 1
i3 — C — Chk^— C1—SCoA

1 1
L- Ch3—C-*—SCoA
HMG-CoA
synthasie 1 CoASH
OH 0
[ I 1
“O-* C— Cbfc—C“CtV“ C—SCoA

ch3
|! hydroxy |!. ne:hy. 'i'Jlaryi Co A HMS Go A

HMG-CoA |
c 2NADFH + 2H'

reductese « * 2NADP*
OH Ir*1 Coash
I I
“O— O— CH?— C — Ona— CHnOH

ch3 H3PO4 CC-? CS 33

, alonate

r+ADr

~ Squalene;

NADP NADPH + H+

Ci 0 — Geranyl pyrophosphate C15 — Famesyl pyrophosphate

Fig. 7.38. The pathway of cholesterol synthesis. C„- — Geranyl pyrophosphate, Cl3 — Farnesyf
pyrophosphate

1 n. the fol low i ng sequence ofreactions, the double bonds in these 5-carbon isoprenes
allow them to condense with the formation of a 10-carbon chain compound, known
as geranyl pyrophosphate. Pyrophosphate groups of these isoprene units are the energy
source for the reaction. Geranyl pyrophosphate then undergoes the condensation
with the third molecule of isopcntenyl pyrophosphate with, the formation of 15 carbon
molecule brnesyl pyrophosphate. After this, two molecules of famesyl pyrophosphate
react, both pyrophosphate groups are removed and 30 carbon, molecule squalene is
formed.
374 Chapter 7. Lipid metaD ol ism

The last stage of the synthesis is the conversion of squalene to die cholesterol
One atom of oxygen is included in squalene by monooxygenase with the formation
of 22-epoxide. Then the linear structure of squalene epoxide is converted to the
cyclic, structure of lanosterol, a sterol with the four-ring structure characteristic of
the steroid nucleus. The sequence of complex reactions then leads to the formation
of cholesterol
Transport of synthesized cholesterol from the liver. Cholesterol synthesized in liver
and del ivered by chylomicron remnants forms cholesterol pool. In the liver cholesterol
is used for the formation of membranes of hepatocytes, for bile acids synthesis and a
fraction of cholesterol is converted to cholesterol esters by acyl-CoA — cholesterol
acyl transferase (ACAT).
Cholesterol leaves the liver by one of the following routes. The hepatocytes package
cholesterol esters together with cholesterol, triacylglyccrols, phospholipids and the
major apoprotein B-100 into nascent very low-density lipoproteins (VLDL). These
particles then arc secreted into the bloodstream by cxocytosis. The nascent VLDLs
acquire apoCll and apoE from circulating high-density lipoproteins (HDL) in the
blood and become the mature VLDL transporting cholesterol and TAG to peripheral
tissues. Another fraction of cholesterol is secreted as bile acidsand biliary cholesterol
to the bile and that is the main route of excessive cholesterol excretion from the liver
(Fig. 7.37).
Regulation of the cholesterol synthesis. In healthy individuals, there is an inverse
relationship between dietary cholesterol intake and the rate of cholesterol biosynthesis.
This regulation ensures a relatively constant daily supply of cholesterol. The rate of
the cholesterol synthesis in the liver is regulated through the key enzyme HMG-CoA
reductase catalyzing the conversion, of H MG-CoA to mevalonate. The regulation, of
HMG-CoA reductase activity is controlled in multiple ways.
Transcriptional control is one of them (Fig. 7.39). HMG-CoA reductase has a life
span of approximately 4 hours and a change in its rate of synthesis or degradation can.
affect cholesterol synthesis rather rapidly that looks like feedback inhibition. When
cholesterol levels in the hepatocytes are high, it binds to the transcription factors that
leads to a decrease in. transcription of the HMG-CoA reductase gene and thus less
enzyme is produced, and less cholesterol is synthesized. When cholesterol level in the
hepatocyte lowers, the rate of transcription increases. Asa result, synthesis of HMG-
CoA reductase increases, and production of cholesterol rises. Transcription of HMG-
CoA reductase gene is also regulated by thyroid hormone acting as inducer and bile
salts repressing the gene.
Proteolytic degradation of HMG-CoA reductase is another mechanism of
regulation. High levels of cholesterol and bile sails in the hepatocytes cause the
conformational changes in the membrane domain of HMG-CoA reductase that
makes the regulatory enzyme more susceptible to the action of proteolytic enzymes
and the rate of cholesterol synthesis decreases as a result.
Covalent modification of HMG-CoA reductase as a result of hormonal regulation.
HMG-CoA reductase activity is regulated by phospho rylai ion/dcphosphorylation
(Fig. 7.40). In lasting state, hormone glucagon stimulates the phosphorylation of the
key enzyme and phosphorylated enzyme becomes inactive. Therefore, in fasting state
7.10. Cholesterol Funciions and Metabolism. Regulation of Cnolesteroi Syntnesis 375

the rate of cholesterol synthesis decreases. Increased levels of intracellular cholesterol

also may stimulate the phosphorylation of HMG-CoA reductase reducing its activity
(feedback suppression).

Fig. 7.39. The regulation of cholesterol synthesis by transcriptional control ot the key enzyme HMG-CoA
reductase, if cholesterol levels in me cell are low SREBP (steroid response element binding protein)
can bind to SCAF1 (SREBP cleavage-activating protein). The SCAP/SREBP complex transfers from the
EFl to me Golgi apparatus, where SREBP is cleaved by a protease, releasing the active transcription
factors (TF), wtiicn are translocated to me nucleus and activate the SRE (steroid response element)
thus inducing me transcription of the HMG-CoA reductase gene and cholesterol synthesis increases,
if cholesterol levels are high it binds to SCAP/SREBP complex and activate binding of this complex
with another protein — insig (insulin induced gene). This large complex cannot be transferred through
membrane of me Golgi apparatus, transcription factors are not released and me gene is repressed;
cholesterol synthesis decreases

in fed state, elevated insulin levels activate the enzyme phosphatase that
stimulates dephospho ryiation of HMG-CoA reductase, it becomes active and the
rate of cholesterol synthesis increases. The enzyme that phosphorylates HMG-CoA
reductase is the cell * energy sensor*. This enzyme is adenosine monophosphate
(AM P)-activated protein kinase (AMPK) which itself is regulated by AMP-activated
protein kinase kinase (AMP is the product of ATP cleavage). Cholesterol synthesis
is highly ATP depended process, thus, cholesterol synthesis decreases when .ATP
levels in the liver cells arc low (AMP level is high) and increases when ATP levels arc
high. All effects regulating the synthesis and the activity of HMG-CoA reductase arc
summarized in Fig. 7.4L
376 Chapter 7. Lipid metaD ol ism

Fig. 7 40 Regulation of HMG-CoA reductase activity in me liver toy covalent moctification

MEVALONATE *- Cholesterol

mRNA

/ndt/crjon Repression
r--------------------- "'i
* Cholesterol
• Bile salts

Gene

Fig. 741. The summary of regulatory effects on HMG-CoA reductase synthesis and activity
7J1. BHe Salts, Functions, Synthesis, ano Role in me Cholesterol Metabolism 377

Insulin/glucagon ratio controls the phosphorylation and dcphosphorylationof the

key enzyme of c hole sterol synthesis. Both glucagon and high AMP level (ATP levels
are low energy slate of the cell is low) cause the H MG-CoA reductase phosphorylation
and thus its inactivation; the rate of cholesterol synthesis lowers. In the fed state, AMP
level is low (AT P levels arc high) and insulin causes de phosphorylal ion of H MG-CoA
reductase; the enzyme becomes active and the rale of cholesterol synthesis increases.
I nsuiin, glucagon and AM P act by covalent modification; statins act as competitive
inhibitors oft he enzyme. The enzyme synthesis is regulated al the level of transcription.
Cholesterol and bile salts cause the repression of the enzyme synthesis and thyroid
hormone acts as inducer.

7.11 BILE SALTS, FUNCTIONS, SYNTHESIS, AND ROLE

IN THE CHOLESTEROL METABOLISM
Cholesterol is the initial precursor for bile salts synthesis, which occurs in the
liver. Approximately 300-500 mg of bile salts arc synthesized daily in the liver. The
bile salts arc:
* needed for emulsification and absorption of dietary lipids, fat soluble vitamins,
particularly vitamin I) from the intestine;
* the end products of cholesterol catabolism excreted from the body;
► the compounds of bile making the cholesterol soluble in the bile by the formation
of micelles.
In the first and regulatory reaction of the bile sails synthesis, the hydroxyl group
al carbon 7 of steroid nucleus is introduced by the 7et-hydroxylase, an ER-associated
monooxygenase found only in the liver (Fig. 7.42). This enzyme requires molecular
oxygen, NAD PH. and cytochrome P450.1 n the next reactions, the double bond in the
B-ring of cholesterol is reduced, the hydroxyl groups arc inserted, at specific positions
on the steroid structure and hydrocarbon chain is shortened by three carbons,
introducing a carboxyl group at the end of the chain. The most common resulting bile
acids arc cholic acid with n-hydroxyl groups at carbons 3,7,12 and chenochotic acid
with a-hydroxyl group at carbons 3 and 7. These bile acids arc named ^primary* bile
acids. Bite acids exist in. their ionized form and usually named bile salts.
Regulation of bile salts synthesis and cholesterol elimination from the body.
Synthesis of the bile salts is regulated through the rate-limiting reaction catalyzed
by 7a-hydroxylase. This reaction is the first on the way of bile salts synthesis from
cholesterol. 7a-hydroxylase synthesis is regulated on the transcriptional level
by cholesterol and thyroid hormones, which both induce the enzyme synthesis,
(Fig. 7.42). Activation of 7a-hydroxylase synthesis results in more active conversion
of cholesterol into the bile salts which arc eliminated with feces and it is a. major route
for the removal of excessive cholesterol from a body Bile salts and estrogens acL as
coreplessors of 7a-hydroxylase gene and thus reduce the bile salts synthesis. These
regulatory effects ensure the normal level of free cholesterol in. the liver and thus in
the body. As thyroid hormones induce the synthesis of 7a-hydroxylasc and in this way
stimulate the cholesterol elimination from a body, patients with, hypothyroidism have
the increased cholesterol levels in blood.
378 Chapter 7. Lipid metaD ol ism

Conjugation of bile salts. The main function of the bile salts is the emulsification
of dietary fats in the lumen of the intestine for their digestion and formation of
mixed micelles required for the absorption of products of digestion. Bile salts arc the
detergents which activity depends on the amphiphilic properties of the molecules.

. ct-hycruxycf olesterol I

Fig. 742. Synthesis of the primary Hile acids — cholic and chenocholic. 7u-hydroxyiase is the key
enzyme cf bite acids synthesis. The synthesis of this enzyme is regulated at transcriptional level.
Cholesterol and thyroid hormone KJ increase the rate of enzyme synthesis and bite salts and
estrogens suppress it
7J1. BHe Salts, Functions, Synthesis, ano Role in me Cholesterol Metabolism 379

Conjugation reaction increases the amphiphilic properties of the bile salts that makes
them better detergents. At first, the carboxyl group al the end of the side chain of
the bile salt is converted to active-CoA derivative and then it is conjugated with
glycine or taurine (Fig. 7.43). Owing to the functional groups of glycine and taurine,
the conjugated bile salts arc more ionized in the contents of the intestinal lumen,
therefore, are more potent detergents. The conjugated bile salts arc laurocholic,
glycocholic, taurochcnocholic and glycochcnocholic. The synthesized bile salts
are secreted into the bile. Bile consists of a mixture of organic and inorganic
compounds. Bile salts and phosphatidylcholine arc the main organic compounds of
the bile. Bile can cither pass directly from the liver where it is synthesized into the
duodenum through the common bile duct or can be stored in the gallbladder when
not immediately needed for digestion. Bile is released from the gallbladder into the
intestine after a fatty meal. The gal I bladder con tract ion is stimulated by the intestinal
hormone cholecystokinin. In the intestine, the bile sails emulsify the dietary lipids
that arc needed for the activity of pancreatic lipase and form mixed micelles for the
absorption. The enzymes of intestinal bacteria cleave off the taurine and glycine from
primary conjugated bile salts and remove a hydroxyl group al position. 7. Asa result,
the primary conjugated bile salts are converted to secondary bile salts — dcoxycholic
and lithocholic (Fig. 7.44). The dcconjugatcd and de hydroxylated bile salts are less
soluble and therefore are less readily absorbed from the intestinal lumen than the
primary conjugated bile salts. About 95% of bile salts secreted to the intestine arc
resorbed and return to the liver via the enterohepatic circulation (Fig. 7.45) but the
bile salts mainly excreted with feces are the secondary bile salts. Only 5% of the bile
salts entering the gut arc excreted with feces every day.
The resorbed secondary bile salts arc rcconjugatcd in the liverbut not hydroxylated
and secreted again into the bile. Because of the enterohepatic circulation, both pri mary
and secondary’ bile salts arc present in the bile. Each bile salt molecule is recycled live
to eight limes every day, and it remains in the system about one week before it is finally
excreted.
Gallstones disease. Cholesterol is the most water-insoluble constituent of the
bite; and to be kept in solution, it must be included into bile micelles. Bile salts
and phosphatidylcholine secreted from the liver into gall bladder serve as detergents
and form these micelles. Cholesterol is soluble in the bile as micelle’s compound
only if the ratio of the bile compounds is 12,5:2,5:1 (Bile salts: Phosphatidylcholine:
Cholesterol). If the secretion of cholesterol into the bile increases, the micelles
arc destructed, cholesterol loses solubility and start to sediment in the gall bladder
forming the stones. The stones consisting of only cholesterol {white cholesterol
stones) or cholesterol aggregated with bilirubin, calcium and other compounds
(mixed brown stones) can be formed in the gall — bladder. The tendency for
cholesterol stones formation is inherited, occurs more frequently in females than
in males, and is associated with obesity. The bile salts c he nod coxy cholate and its
isomer ursodeoxy cholate arc available for oral use to dissolve gallstones at the initial
steps of their formation.
380 Chapter 7. Lipid metaD ol ism

HjN - CH2 - CHs - so3-

rTaunne}

Fig. 7.41 Fomialion of the conjugated primary bile sails

Fig. 7.44. The secondary hile sails formed in the intestine by bacteria I enzymes
7.12. Cholesterol Transport Dy Blood Lipoproteins 381

Gall-bladder

Intestine
circulation

v
Feces (0.2-0.8 g/day)

Fig. 7.45. Enterohepatic circulation of me Pile salts

7.12. CHOLESTEROL TRANSPORT BY BLOOD LIPOPROTEINS

Waler-insoluble cholesterol and cholesterol esters must be transported by blood
packaged in lipoproteins. Free cholesterol molecules are dispersed in the lipoprotein
shell with their hydroxyl group turned outside; cholesterol esters completely
hydrophobic are in the core of the lipoprotein particle. Dietary (exogenous) cholesterol
is packaged in the epithelial cell of the intestine into the chylomicron nascent which
is transferred at first into lymph and then to the bloodstream ( Fig. 7.46). In the blood
nascent chylomicron is converted into the chylomicron mature acquiring apo Cl I
and apoE from HDL. Then, lipoprotein lipase hydrolyzes the triacyl glycerols in the
mature chylomicron and it is converted into chylomicron remnant. Thus, chylomicron
remnants arc avoiding TAG and enriched in. cholesterol and cholesterol esters. These
particles are taken up by the liver through the receptors recognizing apoE and dietary
cholesterol enters the hepatocytes.
Epithelial cells Lymph Stood Liver

Fatty acids to peripheral tissues

Fig. 7.46. Transport of exogenous cholesterol from me intestine to peripheral tissues. C — cholesterol:
CE cholesterol esters; CMn—chylomicron nascent: CMm —cnylomicron mature; CMr — chylomicron
remnants
382 Chapter 7. Lipid metaD ol ism

The endogenous cholesterol synthesized mainly in the liver is released to

bloodstream in nascent VLDL, which major apoprotein is apo 13-100. In blood
nascent VLDL like chylomicron acquires apoCll and apoE from circulating HDL
and is convened to the mature VLDL (Table 7.3). These panicles arc transported
to the peripheral tissues where they undergo the action of LP-lipa.sc. attached to the
capillary walls. LP-lipase activated by apoCll of VLDL hydrolyzes TAG in VLDL
and the content of TAG in the particles decreases as the cholesterol increases. VLDL
are convened into more dense particles enriched by cholesterol — al Hist into IDL
(intermediate density lipoproteins} and then into LDL (low-density lipoproteins)
containing about 50% of cholesterol and cholesterol esters (Fig. 7.47).

Blood HDL

Liver
Fatly acids, glycerol receptors

Fig. 747. Transport of endogenous cholesterol to peripheral tissues. Cholesterol and cholesterol,
esters synthesized in the liver are released to bloodstream in nascent VLDL. in blood nascent VLDL
acquires apoCll and apoE from nigti density lipoproteins (HDL) and are transformed to the mature
VLDL. under the action of LP-lipase VLDL is converted at first into IDL and then into LDL particles
enriched Dy cholesterol. LDL are taken up Uy the celts through special LDL — receptors

Anothcrcnzyme hepatic lipase located in the liver is also involved in the conversion
of IDL into LDL by hydrolyzis of TAG in IDL. When the amount of TAG in the
particles becomes lower apoCll and partly apoE are transferred back to HDL. The
only apoprotein in LDL is a solitary apo 13-100 molecule.
Approximately half of the LDLs arc transported back to the liver, where they are
internalized up by the hepatocytes through the receptor-mediated endoevloses. The
remaining LDL particles deliver cholesterol to the other tissues where it is used for
the membrane synthesis, production of steroid hormones (adrenocortical and gonad
cells) and vitamin D synthesis. Pan of free cholesterol molecules is converted to the
cholesterol esters by the enzyme fatly acyl-CoA: cholesterol acyttransfcrase (ACAT)
in. cytoplasm of the cells using cholesterol as the precursor for the synthesis of steroid
hormones.
LDL is taken up by the cell through the special receptor on the plasma membrane
which recognizes apoi3-IOO and apo L. Besides LDL, this receptor also binds
VLDL, IDL and chylomicron remnants — all panicles containing cholesterol and
its esters. The amount of LDL receptors on the surface of a hepatocyte is about
50 000-70 000. LDL receptor is coded by the gene located on the short arm of
chromosome 19: the gene is very long and contains 18 exons. The protein encoded
by this gene composed of six different regions (Fig. 7.48). The region at the N end is
7.12. Cholesterol Transport Dy Blood Lipoproteins 383

LDL — binding region, transmembrane region of the receptor contains hydrophobic

rad icals of amino acids that allows the receptor io interact with lipid layer of the
membrane. The LDL receptors arc located in specific areas of plasma membranes
known, as coated pits covered with the protein dathrin. After binding of LDL the
receptor — LDL complex invaginates, formsan cndocytic vesicle and enters the cell
through cndocytosis (Fig. 7.49). These vesicles then fuse with lysosomes containing
degradative enzy mes that cleave the compounds of the cndocytic vesicle and free
cholesterol is available for the cell usage. Part of LDL receptors arc returned back to
the plasma membrane and arc able to bind LDL again.
The synthesis of LD L receptors is regulated by the cholesterol levels within the cell.
H igh levels of cholesterol in the cell decrease the rate of transcription of LDL receptors
(downregulation of receptor synthesis) and thus reduce the amount of cholesterol that
can enter these cholesterol-rich cells by receptor mediated cndocytosis. When the
cholesterol levels in the cell reduce the synthesis of cholesterol receptors increases
(upregulation of receptor synthesis). An increased number of receptors results in
increased uptake of LDL cholesterol from the blood and cholesterol levels in the blood
decrease. The number of LDL receptors on the surface of the cells, the ability of the
receptors to bind LDL and the subsequent post-receptor process can be damaged by a
variety of reasons, mutations, for instance, and all of them can lead to accumulation of
LDL cholesterol in the blood (hypercholesterolemia) and premature atherosclerosis.

Fig. 7.48. Structure of LDL receptor

384 Chapter 7. Lipid metaD ol ism

Fig. 749. LDL uptake By the receptor-mediated endacytosis; recycling ol LDL receptors

Other receptors, such as the LDL receptor-related proteins (LRP) and the
macrophage scavenger receptor (SR) have broad specificity. These receptors
can bind and internalized LDL with modified chemical structure. Two types of
macrophage scavenger receptors are known asSR-Al and SR-A2. The main function
of these receptors is to clear the blood from lipoproteins with modified structure.
The macrophages arc usually located near the endothelial surface of vascular
endothelial cell.
Reverse cholesterol transport by the blood lipoproteins. HDL carries out two main
functions. The first, HDL has a unique function as a donor of apo CI I and apo E
(Tig. 7.50} to VLD L and chylomicron. Apo CH is the activator of LP lipase, and apo
E is a ligand for binding of several types of lipoproteins to the LI>L receptor. HDL
particles arc formed as nascent HDLs, mainly by the liver and less in the intestine.
These particles have the discoid shape and contain more variety of apoproteins than
any other lipoproteins (apo Al, apo AIL apo CL apo CH, apo E) and very low levels
of triacylglycerols, cholesterol and cholesterol esters.
7.12. Cholesterol Transport Dy Blood Lipoproteins 395

Nascent HDL contacts in the blood with nascent VLDL nascent chylomicron and
apoproteins Cl I and E arc transferred from HDL to VL.D Land chylomicron which are
converted to mature particles.
The other main function of HDL is to remove excess cholesterol from the
cholesterol — laden cells and to return it to the liver. a process known as ■ reverse
cholesterol transports. The reverse cholesterol transport requires the transfer of
cholesterol from the cell to the HDL particle. Cells contain the protein ABC! (ATP-
bindin* cassette protein I) which uses the ATP energy to move the cholesterol from
the inner layer of the cell membrane to the outer. H DL particles bind to the outer cell
surface through apoproLcin apo Al and the cholesterol di (fuses from the cell membrane
towards the HDL particle. To trap the cholesterol within the HDL core, cholesterol
must be converted to cholesterol ester by the enzyme LCAT (lecithin cholesterol
acyl transferase) attached Lo H DL surface and catalyzing the reaction:

Chol&sterot + Phosphatidylcholine ------- * Cholesterol ester + 2-Lysophosphatidylcholine

(lecithin)

LCAT transfers a laity acid from lecithin (phosphatidylcholine) in the shell

of HDL to a hydroxyl group of cholesterol, convening it to cholesterol ester.
Hydrophobic cholesterol esters sink into the core of HDL particle and cannot
leave HDL. The originally flat HDL becomes spherical mature HDL particle with
hydrophobic core of cholesterol esters. The particle increases in size and is named
HDLr After that HDL transports cholesterol esters and free cholesterol directly to
the liver where HDL binds to specific receptors recognizing apo E. Together with
Lhis way of transport cholesterol and cholesterol esters arc transferred from HDL,
to VLDL by cholesterol ester transfer protein (CLIP). CETP exchanges cholesterol
esters of HDL1 for triacylglyccrols of VLDL and chylomicrons. After this exchange
HDL contains less cholesterol and more triacylglyccrols and is designated as HDL,.
After binding of HDL3 to scavenger receptors of hepatocytes cholesterol esters arc
transferred into the liver. Cholesterol and cholesterol esters transferred from HDL3
to VLDL and chylomicrons arc returned also to the liver as compounds of LDL and
chylomicron remnants (Fig. 7.50).
II DL cholesterol is considered to be «good cholesterol* because HDL picks up the
excess of cholesterol from peripheral tissues including the walls of blood vessels and
increases the reverse transport of cholesterol from the blood vessels to the liver. In the
liver. cholesterol is converted to the bile salts and secreted to the bile together with
cholesterol itself and finally is excreted with feces. LDL cholesterol is considered «bad
cholesterol* because high levels of LDL i n blood lead to hypercholesterolemia and the
d eve lop me nt of aLhcrosclerosi s.
386 Chapter 7. Lipid metaD ol ism

Fig. 7.50. Function of HDL in cholesterol metabolism: the reverse cholesterol transport to the liver
A1, E, Cil — apoproteins; HDLn — HDL nascent; HDL3 — HDL enriched with cholesterol esters.
LCAT — lecithin cholesterol acyf transferase, CE — cnoiesterot esters. C — cholesterol, CETP —
cholesterol ester transfer protein, HTGl — hepatic triacylglycerol lipase, SR — B1 — scavenger
receptor; ABCl — ATP-hinding cassette protein I

7.13. HYPERCHOLESTEROLEMIA. BIOCHEMICAL ASPECTS

OF ATHEROSCLEROSIS. CHOLESTEROL-LOWERING AGENTS
The normal range of cholesterol in blood is 120—200 mg/dl. There is the direct
correlation between high, plasma cholesterol levels (hypcrcholesterotemia), particularly
LDL cholesterol, atherosclerosis and the incidence of death from coronary heart
disease. To estimate the predisposition ofa patient to atherosclerosis, the atherogenic
coefficient is calculated by the formula:

Total cholesterol - HDL cholesterol

HOL cholesterol

The ratio in healthy adult people is <3,5. This coefficient represents the ratio of
LDL cholesterol and HDL cholesterol. The higher is the coefficient, the higher is the
risk of atherosclerosis.
Hypercholesterolemia can result from a variety of genetic and acquired reasons,
all of which may lead to an accumulation of LDL cholesterol in the blood and
atherosclerosis. Familial (genetic) hypercholesterolemia develops when the cells lack
functional LDL receptors (receptor-negative). Familial hypercholesterolemia is the
result of mutations in the gene encoded L DL receptor. This disease is one of the most
common inherited diseases (heicrozygoic — one per 500 people, homozygote — one
per I million people). H etc rozygotes have approximately half of the normal amount
of LDL receptors on. the cell surface. Thus, the total cholesterol levels in blood
increase twice (350-400 mg/dl) because cells cannot take up LDL al a normal rate.
Homozygotes produce almost no LDL receptors and cholesterol levels rise up to 700-
7.13. Hypereno lester oiemia. Biocrtem ieal Aspects of Affler oscie rosis. C n oiest erot-Lo wer i ng Ag ents 387

800 mg/dl). More than 350 types of mutations arc known for LDL receptor gene. The
consequences of mutations depend on the location of the protein damage structure.
If mutation changes the structure of LDL — binding region on N-cnd, the receptor
does not bind LDL, if mutation changes the structure of receptor close to C-end, the
receptor binds LDL but cannot be cndocytosed. The result of these mutations is that
blood levels of LDL (and cholesterol) arc elevated because the cells cannot take up
these panicles at a normal rate. Iking impaired in their ability to acquire cholesterol
from LDL, the liver cells increase the rate of endogenous synthesis of cholesterol.
Cholesterol pool in the body increases together with cholesterol levels in the blood.
These mutations are so common because the heterozygous die from the complications
of accelerated atherosclerosis after the end of the reproductive ago when the mutation
has already been transmitted to their offsprings. Thus, there is not much selection
against them.
Another genetic form of hypercholesterolemia develops in the patients who have
unusual apoprotein (a) in LDL panicles. These particles arc called lipoprotein little
*a* to differentiate them from the apoprotein A found in HDL. The patients having
apoprotein (a) have hypercholesterolemia and high risk of atherosclerosis, coronary
artery disease, myocardial infarction and stroke.
Non-genetic types of hypercholesterolemia are the result of:
* excess consumption of fatty animal meal containing plenty of cholesterol;
► excess consumption of carbo hydrates (hypercaloric food};
► chemical modification of LDL and LDL — receptors by glycation of proteins;
► oxidative modification of LDL (cigarette smoking);
► low levels of HDL.
Excess consumption of cholesterol with food leads to its accumulation in the
cells, and intracellular cholesterol inhibits the replenishment of LDL receptors by
down regulation of their expression. L DL particles arc not taken up by the cells with
the normal rale and circulate in the blood longer increasing the cholesterol levels in
blood. Overeating of carbohydrates also causes hypercholesterolemia, as all substrates
required! for cholesterol synthesis originate from glucose catabolic pathways (acetyl-
CoA, NAD PH and ATP). Excess synthesis of TAG and cholesterol leads to increased
synthesis of VLDLs which arc converted to LDL.
Prolong hyperglycemia in patients with diabetes mcllitus type 2 increases the rate
of noncnzymalic attachment of glucose through NIL group to various proteins in the
body (glycation of proteins). Glycation involves all proteins, including LDL receptors
and apoB-IOOof LDL particles changing their conformation. In healthy people, the
process of proteins glycation occurs at a low rate, but in patients with diabetes me ll it us
it increases. LDL receptors and their ligands become noncomplemcntary and less
circulating LDL arc internalized into the cells through specific receptor — mediated
uptake. That results in more active capture of modified LDL particles by scavenger
receptors of macrophages converting them to «foam cells*. The macrophages
overloaded with cholesterol and modified lipids which arc accumulated in cytoplasm
of the cells arc named «thc foam cells*.
Oxidative modification of LDL particles is a result of the peroxidation of
polyunsaturated fatty acids radicals (Ch. 5) of phospholipids and cholesterol esters
in LDL. Nonenzymalic peroxidation of polyunsaturated fatty acids can be initiated
388 Chapter 7. Lipid metaD ol ism

by free radicals formed during cigarette smoking. Oxi datively modified particles
arc more available for nonspecific uptake of LDL by the macrophage nonspecific
<■ scavenger* receptors. The scavenger receptors arc not down regulated as the LDL
receptors that allow the macrophages to lake up oxidatively modi lied LDL particles
long after intracellular cholesterol levels arc elevated (Fig. 7.51). The formed foam cells
can migrate through the space between endothelial cells and initiate the process of
atherosclerotic plagues formation.

Scaversger receptors

fig.7-51-The foam cells formation. ROS - reactive oxygen species and nitric oxide initiate peroxidation
of polyunsaturated fatty acids the compounds of LDL Vitamin E C and fl-carotene act as antioxidants
and irtfiitiit peroxidation of lipids, thus stabilizing structure of LDL* (modified LDL)

The foam cel Is accumulate in the subindmal space ofarteries and cause atherosclerotic
lesion within the wrall of an artery by the formation of laity streaks. The damaged
endothelial cells start to secrete adhesion molecules that stimulate the circulating
monocytes to accumulate under the endothelium (Fig. 7.52). This movement of
monocytes and macrophages to the injury tissue resembles the inflammatory response.
That loads to suggestion that atherosclerosis is the inflammatory disorder and can be
treated with anti-inflammatory drugs. The deformation of the endothelium exposes the
compounds of the underlying extracellular matrix to the blood and these areas serve as
sites for adhesion and aggregation of platelets. Activated platelets secrete cytokines and
thromboxanes that stimulate this process and increase the risk lor thrombus formation
locally. I f thrombus grows, it can occlude the blood vessel and cause an acute infarction
of the tissue.
Cholesterol-lowering agents. Hypercholesterolemia leads to the development of
atherosclerosis and its complications such as heart attack and stroke. As these are
the most abundant causes of the death, among the people, the very intensive research
develops to prevent and to treat hypcrchole sterol cm iaand atherosclerosis. Healthy food
is important factor in reducing the risk of atherosclerosis. Food of plant origin does
not con rain cholesterol and is enriched writ h native antioxidants such as vitamins E and
C (ascorbic acid), fl-caroLcnc. These vitamins inhibit peroxidation of polyunsaturated
7.13. Hypereno lester oiemia. Biocnem ieal Aspects of Atner oscie rosis. C n oiest erot-Lo wer i ng Ag ents 339

Fig. 7.52. Amero sclerotic plaque formation Oxidatively modified LOL particles are taken up Dy the
macrophage ^scavenger* receptors: tne macrophages overloaded Dy cholesterol are converted to
the foam ceils. These cells are accumulated in su Dendotneii urn. Tne deformation of the endotneiiirm
exposes me compounds of tne underlying extracellular matrix to the Diood and this area serves as site
for adhesion and aggregation of platelets. The risk forinromous formation increases locally

Tatty acids of cholesterol esters of LDL and prevent the formation of the oxidatively
modified LDL particles. Thai reduces the rate of conversion of macrophages to the
foam cells and initiation of the atherosclerotic plaques formation. Fiber, the compound
of whole-grain rye Hour, is not digested in the intestine and absorbs fraction of diclary
cholesterol: then the cholesterol together with fiber is excreted with feces. So low
calorie and low cholesterol food containing native antioxidants and enriched with
fiber is useful for lhe prevention of hypercholesterolemia and atherosclerosis.
* The first step in. treatment of the patient with hypercholesterolemia is a diet
with a low level of cholesterol (<300mg daily). If this diet cannot reduce the
blood cholesterol to the desired levels, the daily cholesterol consumption must
be reduced to <200 mg. Low carbohydrate diet reduces the availability of acctyl-
CoA — the initial substrate for cholesterol synthesis and also lowers the secretion
of insulin activating H MG-CoA reductase.
► The resins, such as cholestyramine and colestipol, which bind the bite salts
within the lumen of intestine and interrupt the cntcrohcpatic circulation, arc
used as cholesterol-lowering drugs. These drugs act as a bile sails sequestra nt
because lhe re sin-bound bile salts arc excreted with the feces and do not recycle
to the liver. This, in turn, stimulates the hepatocytes to uptake more LDL from
the blood because more cholesterol is required for the synthesis of bile salts.
As a consequence, the blood levels of LDL cholesterol arc decreased. The use
of the bile salts scqucstranis cause many gastrointestinal side effects and new
cholesterol absorption blocker is introduced. Ezctimibe reduces the absorption
of dietary cholesterol from the lumen of the gui. The net result is the reduction
390 Chapter 7. Lipid metaD ol ism

of cholesterol pool in hepatocytes. This effect causes the induction of the LDL
receptor synthesis by the liver cells and the capacity of the liver to uptake more
LI>L from the circulation increases. These events lead to lower levels of LDL
cholesterol in the blood.
► The drugs statins the potent inhibitors of HMG-CoA reductase arc used if
the levels of the cholesterol in blood arc very high as in patients with familial
hypercholesterolemia. The statins have the structure similar to the structure of
HMG-CoA — the substrate of the HMG-CoA reductase. Therefore, the statin
binds to the active site of the HMG-CoA reductase and acts as competitive
inhibitor of the key enzy me, thus, reducing the rate of cholesterol synthesis in
the liver cell. Low levels of cholesterol in the cells stimulates the expression of
LDL receptors. As the number of receptors rises on the cell surface, the uptake
of LDL by the cells increases. As a rcsulL, the blood level of LDL cholesterol
decreases.

The quantitative characteristics of the cholesterol metabolism, Blood:

> total cholesterol 120-200 tng/dl;
> LDL cholesterol < 100 mg/dl:
* HI) L cholesterol > 40 mg/d I:
* atherogenic coefficient <3,5.

Review test
L Cholesterol synthesis. Match the figure with the letter.

□
I
CH3— C—S&aA
[Acetyj-CcA] O
I
CHz— C — 5C0A

CaASH

CH3—C*—CH2—
[Aceioacgiyi CaAj; o

CH-3—C—SCoA

CaASH

2NADPH+2H* 2NAD* CaASH

0
SCaA
□CHtOH
7.13. Hypereno lester oiemia. Biocrtem ieal Aspects of Affler oscie rosis. C n oiest erot-Lo wer i ng Ag ents 391

A. Mevalonate.
B. HMG-CoA.
C. HMG-CoA reductase.
D. HMG-CoA synthase.
E. Acetoacclyl-CoA.
2. Bile salts synthesis. Match the figure with the letter.

A. Cholic acid.
B. Glicocholic acid.
C. Taurocholic acid.
D. Liiocholic acid.
E. Deoxycholic acid.

Situational problems
1. 65 years old woman with low physical activity got the excess carbohydrates
(about 400 g daily) for several years. The laboratory value of total cholesterol in
scrum blood was 260 mg/dl. Why the excess consumption of carbohydrates can
cause hypercholesterolemia? To answer the question:
a) compare the cholesterol concentration in the scrum blood of the patient with
the normal range:
b) draw the schemes of the metabolic pathways confirming that overeating of
carbohydrates can result in hypercholesterolemia;
c) draw the scheme of the cholesterol synthesis regulation;
d) give your prescriptions for the diet and treatment of the pau ent.
2. The patient of 48 years old has survived a heart attack. The laboratory lest of his
scrum blood were: total cholesterol 440 mg/dl (reference range is <200 mg/dl).
392 Chapter 7. Lipid metaD ol ism

HDL cholesterol 28 mg/dl (reference range is >40 mg/dl), calculated LDL

cholesterol 340 mg/dl (reference range is < 1.00 mg/dl), triacylglyccrol 160 mg/dl
(reference range is < 150 mg/di). Explain, the results of the laboratory tests and
suggest the possible causes of the patient’s disease. For the answer:
a) draw the scheme of the cholesterol synthesis;
b) draw the formula for the LDL cholesterol calculation:
c) explain the functions of LDL and HDL in cholesterol transport using the
schemes and the results of the laboratory tests:
d) name the most possible cause of the patient’s decease.
3. The gall stones were found in the patient by the upper abdominal ultrasound
investigation. Explain the causes of the gall stones formation. For that:
al describe the composition ofbile and the functions oft he main bile com pounds:
b) draw the scheme of the bile salts synthesis and conjugation:
cl describe the fate of secondary' bile salts and their function in cholesterol
metabolism:
d) explain the way of the gall bladder stones formation.
4. A 50-year-old man with low physical activity used to cat about 250 g of fatty meat
daily for several years. The laboratory value of total cholesterol was 300 mg/dl.
Why can the excess consumption of fatty meat cause hypercholesterolemia? To
answer the question:
al compare, the cholesterol concentration in the scrum blood of the patient with
the normal range;
b) name all possible causes of the hypercholesterolemia;
c) describe the ways of the cholesterol excretion from a body:
d) give your prescriptions for the diet and treatment of the patient.
5. Treatment of the patients with familial hypercholesterolemia by statins
(pravastatin, for instants) allows to decrease cholesterol levels in blood up to the
normal range. Explain the statin action on the cholesterol metabolism. For that:
al draw the scheme of the cholesterol synthesis, ind icate the key enzyme of the
pathway;
b) name all mechanisms of the key enzyme regulation and statin’s action:
cl describe the structure of LDL receptor and its function in cholesterol
metabolism;
d I explain the cause of familial hypercholesterolemia and the symptoms of the
disease.
6. A 60-ycar-old man has been smoking since his 30s. After a heated argument with
his colleagues in office he experienced an acute pain across his chest, associated
with shortness of breath, a sense of light-headedness and sweating. He was
rushed to the hospital emergency departmcni. where his electrocardiogram
showed changes consistent with an acute infarction. Blood was sent to the
labora to ry for biochemical tests, including the total cholesterol. LDL cholesterol
and H DL cholesterol. The blood lipid levels (in mg/dL) were: TAG — 290,
total cholesterol — 325, LDL cholesterol — 245, HDL cholesterol — 25. The
patient was treated intensively and got better. Before he was discharged from the
7.13. Hypereno lester oiemia. Biocrtem ieal Aspects of Affler oscie rosis. C n oiest erot-Lo wer i ng Ag ents 393

hospital his physician told him that his in fare Lion is the result of a long history
of smoking. Explain the conclusion of the specialist. For that:
a) explain the functions of lipoproteins in cholesterol transport using the
schemes:
bi describe the structure of LDL receptor, regulation of its synthesis in the liver
and function in. cholesterol metabolism;
cl name all possible causes of the hypercholesterolemia;
d) explain the mechanism of prolonged smoking on. the hypercholesterolemia
and atherosclerosis development.
7. LDL/HDL ratio is important for the regulation of cholesterol levels in
blood. Disbalance of these lipoproteins leads to hypercholesterolemia and
atherosclerosis development. What are the functions of these lipoproteins in
cholesterol metabolism? For the answer draw:
a) the scheme, representing LDL function in cholesterol metabolism;
b) the scheme, representing HDL function in cholesterol metabolism;
c) the reaction catalyzed by LCAT and explain the role of this reaction in HDL
functioning;
d) the formula for the atherogenic coefficient and indicate its normal range.
8. Hypercholesterolemia is a frequent complication of diabetes mellitus in patients
with prolonged hyperglycemia. Why a high level of glucose in blood causes
hypercholesterolemia and atherosclerosis? For the answer explain:
a) how glucose can interact with the proteins and the consequences of this
reaction for the proteins;
b) glycation of which proteins results in hypercholesterolemia;
c) possible causes and complications of hypercholesterolemia.
9. 65 years old man, who considered him self healthy was examined prophy tactically.
The laboratory value of total cholesterol was 240 mg/dl The atherogenic
coefficient was 3.8. A .specialist prescribed to increase physical activity and
correct food composition. He was advised to decrease the red meat consumption
and to change his diet for vegetables, enriched with cellulose vitamins C and E.
Explain the specialist's prescriptions. For that:
a) name all causes of acquired hypercholesterolemia:
b) explain, why the red meat must be excluded from the diet of the patient;
c) explain, why a diet enriched with cellulose vitamins C and E can influence on
the cholesterol levels in blood;
d) explain the formation of the foam cells and their role in atherosclerosis
development.
 Chapter 8
NITROGEN METABOLISM

8.1 Digestion of Proteins and Amino Acid Absorption. Essential Amino Acids
82. Transamination and Deamination of Amino Acids
8.1 Ammonia Detoxification. Urea Cycle. Urea Cycle Disorders, Hyperammonemia
8.4. Synthesis of Nonessential Amino Acids
8.5. The Main Steps of Amino Acid Degradation; Glucogenic, Ketogenicand both Gluco
and Ketogenic Amino Acids
8.6. Special Pathways of Some Amino acids: Phenylalanine Metabolism, Tetra hydrofolate,
Vitamin B^and s Adenosylmethionine
8.7. Decarboxylation of Some Amino Acids, Synthsis of Neurotransmitters and Hormones
8J. Special Products Derived from Amino Acids

8.1 DIGESTION OF PROTEINS AND AMINO ACID ABSORPTION.

ESSENTIAL AMI NO ACIDS
Major proteins of the body arc constantly degraded Lip to amino acids and
resynthesized. The assembly of new proteins requires a source of amino acids.
These building blocks arc generated by the digestion of the dietary proteins in the
gastrointestinal tract and the degradation of proteins within the cell.
Amino acids perform the following functions in the body:
► anabolic: in addition to their role as the ami no acids arc
precursors of many specialized bio mol ecu les, including hormones, coenzymes,
nucleotides, alkaloids, cell wall polymers, porphyrins, antibiotics, pigments, and
neurotransmitters;
► energetic: amino acids arc the source of energy under extreme conditions (during
starvation or in uncontrolled diabetes mellilus, when carbohydrates arc either
unavailable or not properly utilized , cellular proteins arc used as fuel).
Proteins cannot be stored in the body and don’t form any tissue depot such as
glucose (in the form of glycogen) or fatty acids (as TAG). That is why the amino
acid reserve is mainly muscular and blood plasma proteins. In human body,
approximately 400 g of proteins arc degraded and the same amount synthesized
everyday. Amino acids that arc not incorporated into a new protein and unnecessary
for immediate requirements canT be stored and undergo oxidative degradation.
One important feature distinguishes amino acid degradation from other catabolic
processes described to this point: every amino acid contains an amino group, and
the pathways for amino acid degradation, therefore, include a key step in. which the
8.1. Digestion of Proteins ana Amino Acid Absorption. Essential Amino Acids 395

a-amino group is separated from the carbon skeleton and shunted into the pathways
of amino group metabolism.
zl/l 20zfm/no acids present in linntan body profems arc c/assi/ied info 4 groups:
► non-essential amino acids which are synthesized in the body in the adequate
amount: Ala, Asp, Asn, Glu, Gin. Pro, Gly, Ser;
► essential amino acids which are not synthesized in the body: Vai, Leu., lie. Met,
Phe, Trp, Lys, Thr;
► partially essential amino acids which arc synthesized i n insufficient quantities for
body needs: Arg, His;
► conditionally essential amino acids which arc synthesized from essential amino
acids: Cys. Tyr (from Met and Phe).
The quantities of aig/nine generated by the urea cycle (see below) arc adequate only
for the adult and arc insufficient to support growth.

Nitrogen balance
Amino acids include 95% of all body nitrogen. The organism's nitrogen balance
is therefore primarily determined by protein metabolism. Attrogen balance is tke
correlafion between nitrogen intake and nitrogen lost Ay tAe Aody. Nitrogen is lost
mainly as urea in the urine, as well as in feces and saliva, by desquamated skin, hair,
and nails.
hi adults, the nitrogen balance is generally in equilibrium — i.e., the quantity of
protein nitrogen taken in and excreted per 24h is approximately equal.
I f some of the nitrogen taken in is more than tost, then the balance is postfive. This
is the ease duri ng growth. pregnancy, tissue repair after injury, recovery from illness: in
other wo rds, the situations when the protein synthesis exceeds the protein degradation.
A^arire balances (if nitrogen taken in is less than lost) arc rare and usually occur due
to disease: tuberculosis, cancer: negative balance occurs in starvation and may be in
very old individuals.
Lost cofjrcifn; is quantitatively determined by protein degraded in the complete
protein starvation. So under the condition of complete protein starvation 25 g of tissue
proteins arc degraded in 24 h.
Protein minimum is determined by the amount of protein needs for nitrogen
equilibrium, it is 30-50 g (-40 g) of proteins per day. But this quantity is not enough
for physical activity, capacity for work and health.
Protein optimum is determined by the amount of protein necessary for normal
growth and development: this quantity is 100-120 g per day for adult.
Anfririotts (alimeataryj value of protein depends on amino acid composition
and ability for assimilation: if protein contains the essential amino acids in. the right
proportion required by humans, it has AigA biological va&e.
Animal proteins arc high-value ones: they contain essential amino acids in
adequate quantity and proportion, and arc more digestible than plant proteins which
are low-value (Table 8.1). Plant proteins generally have a lower content of some
essential amino acids, such as lysine, tryptophane and methionine. Soy protein is
one of the best plant proteins, but nevertheless, the most prominent difference is
the proportion of the essential sulfur-containing amino acid methionine. Egg white
396 Chapter 8. Nitrogen metabolism

protein has approximately three limes more methionine than is found in soy protein.
Vegetable proteins arc usually badly digested and some plants (for example, legume
seeds and cereals) involve protease inhibitors, which arc peptides that form stable
inactive complexes with some of the pancreatic enzymes causing protein maWiges/jon.
Protein-energy malnutrition can lead to severe disorders occurring in growth
children, usually under 5 years old of age in developing areas of Asia, Africa, and
South America. Two extreme forms arc recognized: marasmus and Jbrasftjr&rAor. In
kwashiorkor energy intake may be adequate, but there is a deficiency in both the
quantity and the quality of protein. Kwashiorkor is characterized by edema and swollen
belly (as a result of low levels of proteins in blood), liver enlargement, the change in
skin and hair color Ito a rust color, caused but insufficient synthesis of melanins),
fatigue, diarrhea, loss of muscle mass, damaged immune system, which can lead to
more frequent and severe infections, and irritability. Nutrient deficiency is the main
cause of marasmus. Marasmus is a state of extreme emaciation; it is the outcome of
prolonged negative energy balance. It occurs in children who don't ingest enough
calories, proteins, carbohydrates, and other important nutrients. The main symptom
of marasmus is underweight. Children with this condition have lost a lot of muscle
mass and subcutaneous fat and stunted growth. They suffer from chronic diarrhea,
respiratory infections, and intellectual disability.
Table 8.1. Biological value and Uigeslibilily of food proteins

Food Biotoqrcat Value

Digs STibiliiy oT Protein
Eggs (whole) too
ChrekerVTurttey 79 T r
Beef 78
Fish 70
Brown rice 57
Peas 55
—
Whole Wheat 49 __ —■ — -
Soy deans
Com
47
36
L .
Egg Ciiiken F<sh Aice WJiole Coen Wheat
Dry Beans 34 maal mesi wiea gluten

The Biological Value (8V) is a scale of measurement used to determine what percentage
of a given nutrient source is utilized by the body: in short — BV refers to how well and
how quickly your body can actually use the protein you consume). The highest BV of any
food source is 100%. Egg white protein is considered to have one of the best amino acids
profiles for human nutrition

Certain adult people in developed countries arc also al risk. This also includes
vegetarians and vegans who follow an imbalanced diet, as well as institutionalized
older people and hospitalized patients.
Some individuals experience distress when eating gluten-containing foods, and it
is commonly referred to as gluten sensitivity (GS> or gluten intolerance. Gluten is
a family of proteins found grains like wheal, rye, spelt and barley; it is protein that
grain plants use to store nutrients for the next generation of plants. Gluten intolerance
8.1. Digestion of Proteins ana Amino Acid Absorption. Essential Amino Acids 397

estimates between 0.5 and B percent of the population. Symptoms which occur
shortly after consuming gluten involve diarrhea and constipation, bloating, abdominal
pain, nausea and fatigue. People who have gluten intolerance have to avoid any food
with gluten in it, which includes any food that contains: wheat, and any derivatives of
wheat, barley, rye, brewer’s yeast (that is usually derived from beer): pasta, bread and
pastries, many baked goods, noodles, crackers, cereals, pancakes, waffles, and crepes,
biscuits etc.

Protein digestion
In humans, the degradation, of ingested proteins to their constituent amino acids
occurs in the gastrointestinal tract. Enzymes participating in protein digestion arc
proteinases (the same — peptidases, proteases, proteolytic enzymes), which mostly
released to gastro-intestinal tract in. inactive form (zymogens) and activated by partial
proteolysis in the lumen of stomach and intestine. Activated peptidases specifically
hydrolyze peptide bonds formed by appropriate amino acids. Endopeptidases
(c.g., — pepsin, trypsin) cleave the internal peptide bonds in the polypeptide chain;
exopeptidases (e.g., — carboxypeptidase, aminopeptidase) remove either N- or
C-terminal amino acid (Fig. 8.1).

Fig. 8.1. Digestion of proteins in the gastrointestinal trad. Marked in blue proenzymes are inactive
precursors of pepsin, trypsin, enymotrypsin, elastase and carboxypeptidase
398 Chapter 8. Nitrogen metabolism

Proteins arc not digested in the oral cavity. The entry of dietary proteins into the
j/owtic/j stimulates the gastric mucosa to secrete the hormone gastrin, which in turn
stimulates the secretion of hydrochloric acid by the parietal cells and pepsinogen by
the chief cells of the gastric glands. The acidic gast ric juice (pH 1.0 to 2.5) is both,
.antiseptic, killing most bacteria and other foreign cells, and a denaturing agent,
unfolding globular proteins and rendering their internal peptide bonds more accessible
to enzymatic hydrolyzis. An acidic medium is important for pepsin, which optimum
pH activity spans within 1-2. Pepsinogen (Afr 40,554), an inactive precursor, or
zymogen, is convened to active pepsin C.Wr 34,614) by the HCI and enzymatic action
of pepsin itself (autocatalysis), the activation occurs by partial proteolysis (Fig. 8.2).

Pepsin gen -peptide Pepsin

(40554)

(Autoactivation)

inactive zymogen? Active enzyme:

pepsinogen pepsin

Masking
sequence

Fig. 8_2. Actrvalion ot pepsin by partial proteolysis

In the stomach, pepsin hydrolyzes ingested proteins at peptide bonds on. the
carboxy]-terminal side of the oromaric a/n/m? ac/J residues (Phe. Trp, and Tyr), as well
as peptide bonds between Glu or Asp with any amino acid, cleaving long polypeptide
chains into a mixture of smaller peptides.
Intrinsic factor is a glycoprotein secreted by parietal cells of the gastric mucosa.
In humans, it has an important role in the absorption of vitamin B]2 (cobalamin) in.
the intestine, and failure to produce intrinsic (actor results in the condition

Lactic acid is not present in stomach juice. But lactate may be formed in the reduced
or complete absence of free hydrochloric acid as a result of accelerated reproduction of
Lactobariffus, or in malignant tumors of the stomach.
8.1. Digestion of Proteins ana Amino Acid Absorption. Essential Amino Acids 399

HCI and pepsin arc able to damage gastric epithelium cells. In healthy people, it
docs not occur due to the presence of protective factors of the mucus tunic, such as:
► formation of mucus on the surface composed of hctcropoiysaccharides which
can't be split by peptidases;
► secretion of HCO^ by epithelial cells creating the less aggressive medium at
parietal layer with pH 5.0-6.0 in which pepsin is inactive;
► besides, damaged epithelium cells possess the ability to rapid regeneration.
The digestion of proteins now continues in the small intestine. The arrival of
amino acids into the upper part of the intestine (duodenum) causes the release of
the hormone cholecystokinin into the blood, which stimulates the secretion of several
pancreatic enzymes with activity optima at pH 7 to 8.
Trypsinogen, chymotrypsinogen, procarboxypeptidases A and B. and proelastase
arc the zymogens of trypsin, chymotrypsin, carboxy peptidases A and B. and elastase
which synthesized and secreted by the exocrine cells of the pancreas. Trypsinogen
is converted to its active form, trypsin by enteropeptidase, a proteolytic enzyme
secreted by intestinal cells. Free trypsin then catalyzes the conversion of additional
try psinogen to trypsin. Trypsin also activates chymotrypsinogen, procarboxypeptidases,
and proelastase tFig. 8.3; 8.4). All activations occur by partial proteolysis (splitting-ofT
peptide from zymogen, more often from N-termini, with rearrangement of the protein
structure that completes the active site turning inactive proteins into active ones).
The synthesis of the enzymes as inactive precursors prevents the cleavage of
ow n proteins of the stomach and intestinal mucosa, and exocrine cells of pancreas
from destructive proteolytic attack. The pancreas further protects itself against self­
digestion by making a specific inhibitor, a protein called pancreatic trypsin inhibitor
that effectively prevents premature production of active proteolytic enzymes within
the pancreatic cells. Stomach ulcers are a type of peptic ulcer disease when pepsinogen

Trypsinogen

Procarboxy Carboxy­
Praelastase Elastase
peptidase peptidase
- - —

/-----------“---------- i
Chymotry-
Chymotrypsin Prolipase Lipase
psinogen

Fig. 8.3. Pancreatic proteinases activation

400 Chapter 8. Nitrogen metabolism

is convened io pepsin within cells of mucous coat of stomach. Acute pancreatitis is

a disease caused by inflammation and obstruction of the normal pathway by which
pancreatic secretions enter the intestine (c.g., — gallstones disease, heavy alcohol
intake). The zymogens of the proteolytic enzymes arc converted to their catalytically
active forms prematurely, inside the pancreatic cells, and attack the pancreatic tissue
itself. This causes excruciating pain and damage to the organ that can prove fatal.
Trypsin, and chymotrypsin further hydrolyze the peptides that arc produced by
pepsin in the stomach. This stage of protein digestion is accomplished very efficiently,
because pepsin, trypsin, and chymotrypsin have different amino acid specificities
(Fig. 8.4). Trypsin cleaves bonds formed by Lys and Arg, chymotrypsin cleaves bonds
formed by Phe, Tyr, and Trp.

Fig. 8.4. Action of the digestive proteases. Pepsin, trypsin, chymotrypsin, and elastase are
endopeptidases; they hydrotyze peptide bonds within chains. The others are exopeptidases;
aminopeptidases remove the amino acid at the N-terminius and the carbaxypeptitfases remove the
amino acid at the C-terminus. For each proteolytic enzyme, the amino acid residues involved in the
peptide bond that is cleaved are listed beside the R group to the right of the enzyme name
8.1. Digestion of Proteins ana Amino Acid Absorption. Essential Amino Acids 401

Carboxypeptidases A and B (both of which arc zine-containing enzymes), remove

successive carboxyl-terminal residues from peptides. Carboxypeptidase A removes
C-terminal amino acids with aromatic and hydrophobic radicals: carboxypeptidase B
removes C-terminal Lys and Arg. Elastase has a rather broad specificity and attacks
bonds next to small amino acid residues, such as glycine, alanine, and serine.
Degradation of the short peptides in the small intestine is then completed by other
intestinal peptidases which arc released in active form by the intestinal mucosa glands.
Aminopeptidase hydrolyzes successive ami no-terminal residues from short peptides.
Dipeptidases cleave di peptides.
Under normal circumstances, the dietary proteins arc almost completely digested
to their constituent amino acids, and these end products of protein digestion are then
rapidly absorbed from the intestine into the portal blood.
There are several different amino acid transporters, with specificity for the nature
of the amino acid side chain (large or small; neutral, acidic, or basic). The various
amino acids carried by any transporter compete with each other for absorption and
tissue uptake. Il is known//w j/jccj/zc frafls/wrf systems providing amino acid transfer
across membranes:
► for neutral amino acids with a short side chain (alanine, serine, and glycine):
► for neutral with a tong or branched side chain (valine, leucine, and isoleucine);
* for cationic amino acids (lysine and arginine);
* for anionic amino acids (glutamic and aspartic acids);
► for imino acids (proline and oxoprolinc).
Free amino acids from the first and fifth groups, as well as methionine arc absorbed
across the intestinal mucosa (Fig. 8.5).
Intestinal
lumen
Ammo

Portal vein

Fig. 8.5. Trans epitnelial amino acid transport Sodium-dependent carriers transport both Na1 and an
amino acid into tne intestinal epitnefial cell from me intestinal lumen. Na* is pumped out on the serosal
side {across tne nasoiaterai membrane) in exchange of by tne Nai,K'-ATPase. On me serosal side,
the amino acid is carried by a facilitated transporter down its concentration gradient into tne blood.
This process is an example of secondary active transport
402 Chapter 8. Nitrogen metabolism

Dipcptidcs and tripeptides enter the brush border of die intestinal mucosal cells,
where they arc hydrolyzed to free amino acids, which arc then transported into the
hepatic portal vein. Relatively large peptides maybe absorbed intact, cither by uptake
into mucosal epithelial cells (tra.nsccll.ular). Many such peptides arc large enough to
stimulate antibody formation. - this is the basis of allergic reactions to food compounds.
y-Glutamyl cycle (Fig. 8.6). y-Giuiamyt cyc/e functions in inlenstine, kidney,
pancreas, liver and spleen: but brain and other tissues do not contain a high
quantity of this system. y-Glutamyl transpeptidase (y-GT) plays the key role in the
system. This enzyme catalyzes the transfer of y-glutamyl group from glutathione
(y-glutamylccsteinylglycinc) or another y-glutamyl peptide to transported amino
acid andthc following transport of the complex into the cell. Glutathione is found in.
all animat tissues. For a single amino acid transport into the cell by y-glutamyl cycle,
three ATP molecules are expended.

Fig 8.6. y-Glutamyl cycle, in cells at tne intestine and kidney, amino acids can be transported across
me cell membrane by reacting with giutatnione ij-giutamyi-cysteinyi-giycine) to form ay-glutamyl
amino acid. The amino acid is released into tne cell, and glutathione is resynthesized. However,
the major role of inis cycle is glutathione syntnesis, and many tissues lack tne transpeptidase and
5-oxoprotinase activities

y-GT activity in blood scrum is 30-50 ME/L (pMol/min* mg) for men and 25 —
35 ME/L for women. Determination of y-GT activity in blood scrum is used for
diagnosis of liver and heart disorders, as well as it is the marker in cancer of pancreas,
liver and prostatic gland. The test is also may be used to reveal persons who at risk of
alcoholism and for control of the treatment of chronic alcoholics.
8.2 Transamination ana Deamination of Amino Acids 403

82 TRANSAMINATION AND DEAMINATION OF AMINO ACIDS

Tissue ami no acids arc derived from both, dietary proteins digested in gastrointestinal
tract and tissue proteins, which arc degraded by lysosomal proteases referred to as
cathepsins (from the Greek «kathcpscin* which means *to digest*). Cathepsins are
distinguished by the structure of their active site, for example, the serine proteases
cathepsins A and G, the aspartic proteases cathepsins D and E, and the cysteine
cathepsins; cathepsins are active in a slightly acidic environment.
One important feature distinguishes amino acid degradation from other catabolic
processes that every' amino acid contains an amino group, and the pathways for amino
acid degradation, therefore, include a key step in which the a-a mi no group is separated
from the carbon skeleton. The ammonia formed in the mitochondria of hepatocytes
is converted to urea in the urea cycle. Urea, production occurs almost exclusively in
the liver and is the fate of most of the ammonia channeled there. The urea passes into
the bloodstream and thus to the kidneys and is excreted into the urine. Deprived of
nitrogen amino acid carbon skeletons arc channeled to gluconeogenesis, ketogenesis,
or degraded to carbon dioxide and water.

Transamination reactions
The first step in the catabolism of most L-amino acids is the removal of the
a-amino groups, catalyzed by enzymes called aminotransferases or transaminases.
During transamination, the a-amino group of an. amino acid is transferred to
a-carbon of ci-ketoacid (Fig. 8.7).

COO’ COO"
l
c=o COO HgN*— C— H coo-
1 I
ch2 + HgbT—C — H ■ b. ch2 + C—O
1 ArrancMr an st erase I
ch2 R tPLP) R

COO" COO"

ci-KetogJutarate L-Amlno acid L-Ckitamate n-Kelo acid

Fig. 8.7. Transamination reactioii The amino group from one amino acid is transferred to anomer. Pairs
of amino acids and tneir corresponding u-keto acids are involved in these reactions. u-Ketoglutarate
and glutamate are usually one of the pairs. Tne reactions, which are readiEy reversible, use pyridoxal
priospnaie (PIP) as a cofactor. Tne enzymes are called transaminases or aminotransferases

There is no net deamination (loss of amino groups) in those reactions, because

the a-kctogluiarate (pyruvate. oxaloacctatc) becomes aminated as the a-amino acid
is deaminated.
Celts contain more than 10 d ill ere nt types of aminotransferases which arc
distinguished by different substrate specificity. Many are specific for a-kctoglutaratc
as the amino group acceptor but differ in their specificity for the L-amino acid. The
enzymes arc named for the amino group donor (alanine aminotransferase, aspartate
404 Chapter 8. Nitrogen metabolism

aminotransferase, for example). The coenzyme pyridoxal phosphate (PLP) is present

at the catalytic site of aminotransferases. PLP is a derivative of vitamin U,. The
reactions catalyzed by aminotransferases are freely reversible.
Nearly all amino acids can enter transamination with the exception of lysine,
threonine, and proline.
TAc biolqgica/ importance a/ frnnsamination reactions:
► the transa mi nation reaction is the first in. catabolic pathway for majority ofamino
acidsand the last in the metabolic way of non-essential amino acid synthesis. As
a result of this type of reaction the redistribution of amine nitrogen occurs in the
body;
► the transamination provides the link of amino acid metabolism with TCA where
a-ketoacidsare oxidized, or they arc converted to glucose or ketone bodies which
are then oxidized;
* amino-group is removed from amino acid without releasing of free ammonia
which is toxic.
Analyses of certain enzyme activities in blood serum give valuable diagnostic
information for a number of disease conditions. Alanine aminotransferase (ALT: also
called glutamate-pyruvate transaminase, G PT) and aspartate aminotransferase (AST:
also called glutamate-oxaloaceiatc transaminase. GOT) (Fig. 8.8) arc important in the
diagnosis of heart and liver damage, drug toxicity, or infection,

COO" COO"

Ok CHg CHq CH2

1 |
HC — NHf C—O + ch?
| ALT i
COO" 0—0 (PLP) COO" HC- NH3'
1
COOT COO"

Alanine n-Ketoglutarate Pyruvate Glutamate

coo- coo- COO" COO"

| i | 1
CHs CH-. CH? CHn
I 1 1 1
HG- NH/ C—O + CH?
| AST 1
COO" C—O (PLP) COO" HC’NHi

COO" COO"

Aspartate n-Ketoglutarate Oxaloacsate Glutamate

Fig. 8.8. Alanine aim aspartate transamination Dy alanine transaminase (ALT) ano aspartate
transaminase (AST)

After a heart attack, a variety of enzymes, including those aminotransferases, leak

from the injured heart celts into the bloodstream. Measurements of the blood scrum
activities (in U/L) of the two aminotransferases by the SGPT (scrum glutamate­
8.2 Transamination ana Deamination of Amino Acids 405

pyruvate transaminase) and SCOT (serum glutamateoxaloacetatc transaminase) tests

arc widely used (Fig. 8.9).

Fig. 8.9. Serum transaminase activity (AST and ALT) in myocardial infarction

The SGOT(AST) and SGPT(ALT) tests arc also important in occupational

medicine, to determine whether people exposed to carbon tetrachloride, chloroform,
or other industrial solvents have suffered liver damage (Fig. 8.10). Liver degeneration
caused by these solvents is accompanied by leakage of various enzymes from injured
hepatocytes into the blood. Aminotransferases arc most useful in the monitoring of
people exposed to these chemicals, because these enzyme activities arc high in the
liver and can be detected in very small amounts.

u/L

Norm

Fig. 8.10. Seram transaminase activity (AST and ALT) in hepatitis

Ritz coefficient is the ratio of AST. ALT in blood serum (11.33 ± 0.42). ALT is
predominantly localized in the cytosol of the liver cells and cardiomyocytes. AST
is found in the cytosolic and mitochondrial fraction of the liver, cardiac muscle
and the majority of organs. Increased activity of mitochondrial AST is found in
acute liver damage and myocardial infarction accompanied by necrosis and cell
membrane distraction.
406 Chapter 8. Nitrogen metabolism

In viral hepatitis (see above Fig. 8.10) the level of ALT increases up to 600—
800 unit/L (5-30 uniis/L in the normal range); AST elevates till 300—400 unit/L (8-
40 nnits/L in the normal range). Thus, the Ritz ratio decreases to 0,6 in hepatitis. In
myocardial infarction (sec above Fig. 8.9), AST increases to 400—500. buL ALT — till
150-200, so the coefficient increases.
Ritz ratio is very’ important for not only diagnosis but to carry differential
diagnostics, for estimation of the effect of treatment and to make a prognosis.

Deamination of amino acids

If the NH.-group is released as ammonia, the process is referred to deamination
reaction. All amino acids arc deaminated, with the exception of Lysine.
There are several types of amino acid deamination in humans.
Glutamic acid oxidative deamination. The glutamic acid undergoes direct oxidative
deamination. In hepatocytes, glutamate is transported from the cytosol into
mitochondria, where oxidative deamination catalyzed by L-glutamate dehydrogenase
(GDH) the most active enzyme takes place (Fig. 8.11). GDH is an oligomer with
6 subunits.
Reaction occurs in two stages: enzymatic dehydrogenation, and then non-
enzymatic hydrolytic cleavage of amino group in the form of ammonia. This reaction,
is readily reversible and can use cither NAD or NAD Pas a coenzyme. The oxygen on
a-ketogluEaratc is derived from H,O.

coo- COO- COO-

1
CH NHg NAD’’ NADH + H* C NH c—o
| nh; 1
ch2 ch2 +h2o ch2
1 ■
Glutamate DH
ch2 ch2 ch2

coo- COO- COO-

Glutamic acid n-Keloglutaric acid

Fig. 8.11. Oxidative deamination of glutamate

Intramolecular deamination of histidine, Hisiidasc-catalyzed deamination of

histidine produces urocanic acid in the liver;

CH2—CH—COO nh;

Histidase
NHg

Histidine Urocanic acid

8.2 Transamination ana Deamination of Amino Acids 407

Histidinemia caused by impaired conversion of histidine to urocan ic acid via

the histidase enzyme is inherited autosomal disorder. Hist id incm ia is defined
biochemically as elevated histidine in blood, urine, and cerebrospinal fluid, and
decreased levels of the urocan ic metabolite acid in the blood, urine, and, skin. In
most individuals with hisiidincmia, the condition is clinically silent and considered
benign, with no need for treatment or a specific diet. In a small subset of patients with
specific events in the neonatal period, such as tow oxygen, it has been suggested that
hisiidincmia may contribute to development of intellectual disability, behavioral or
learning disorders.
Non-oxidative deamination of Ser and Thr. Serine is converted to pyruvate;
threonine — to a-ketoglutarate (by serine dehydratase and threonine dehydratase
respectively, which coenzyme is PLP):

IMHj OH q NH
] 1 1
HO—CHa--CH—COO" CH3—CH--CH—COO"

L PLP L PLP
h2o H20
Serine ^-HsO Threonine ^-HsO
dehydratase dehydratase

^nh;
nh;

o 0

CHa—C—COO' ch3— ch2--C —COO"

Glutamic acid a-Ketobutyrate

Indirect deamination
Most amino acids arc unable to be deaminated in one stage as glutamic acid
(L-a mi no acid oxidases present in tissues have insufficient activity). Amino groups of
these amino acids arc transferred to cr-kctoglutaratc with glutamate formation, which
then undergoes direct oxidative deamination. The mechanism of deamination in twro
stages is referred to as indirect deamination (Fig. 8.12).
Indirect deamination occurs with the participation of two enzymes: aminotransferase
(coenzyme pyridoxvl pyrophosphate) and glutamate dehydrogenase ( coenzyme NAD+).
Indirect deamination is the main pathway ofdeamination of the majority of amino acids.
The effect of transamination reactions is to collect the amino groups from many
di fie rent amino acids in the form of L-glutamate. The glutamate then functions as an
amino group donor for excretion pathways that lead to the elimination of nitrogenous
waste products. Ammonia formed as a result of deamination reactions has to be
transported to the liver for the following detoxification.
Another type of amino acid deamination is indirect nonoxidative deamination
with IMP-AMP cycle characteristic for muscular tissue and brain, where glutamate
408 Chapter 8. Nitrogen metabolism

COO"
I

ci-Kelo add n
Glutamate

Fig. 8.12. The general scheme of indirect amino acid deamination

de hyd rogc nasc has lower activ ily (F i,g. 8.13). A m i no g roup of amino aci ds is t rans ferred
to i M P (inosine monophosphate) by two consecutive reactions of transamination with
the formation of AMP. which is deaminated hydrolytically with ammonia releasing.

Fig. 8.13. Indirect nonoxidative deamination via IMP-AMP cycle

8.2 Transamination ana Deamination of Amino Acids 409

Nitrogen-free residue of amino acid is a-kclo acid which may be involved into:
► oxidation reactions to CO2and H2O;
► transamination reaction lor non-essential amino acids synthesis:
» anap I erotic reactions to* supply TCA ITom the loss of metabolites or other
substances synthesis;
* gluconeogenesis;
► ketogenesis.

The quantitative characteristics of the metabolism of amino acids learnt in this section
Y-GT activity in blood serum:
Men - 30-50 U/L
Women — 25—35 U/L
ALT and AST activity sei blood scrum: 5-40 U/L
Ritz ratio: 1.33+0.42

Review tests
I. Organs, involved into digestion, secret enzymes:

Stomach pH Food
1.5-2.0 proteins

I®
Polypeptides

Small intestine Pancreas

pH 8.0
Oligopeptides
and amino acids
Enterocytos

---------------- Amino acids

Absorption

Enzymes:
A. Aminopeptidase.
B. Trypsin.
C. Amylase.
D. Lipase.
E. Pepsin.
410 Chapter 8. Nitrogen metabolism

2. To digest proteins, the following enzymes are synthesized in cells of stomach, pancreas
and small intestine:

Stomach pF - Food
1.5-2.0 proteins

I®
Polypeptides

Small intestine Pancreas

pH e.o
Oligopeptides
and amino acids
Enterocytos

■ Amino acjds
Absorption

Compounds:
A. Dipeptidase.
B. Chemotrypsin.
C. Lipase.
D. HCI.
E. Amylase.

Situational problems
1. Kwashiorkor, also known as ^edematous malnutrition* because of its association
with edema (fluid retention), is a nutritional disorder most often seen in
regions experiencing famine, h’s most common in sub-Saharan Africa and
other countries where people routinely have a. limited supply of food. It is a
form of malnutrition caused by a lack of protein in the diet. People who have
kwashiorkor typically have an extremely emaciated appearance in all body parts
except their ankles, feet, and belly, which swell with fluid. The symptoms of
kwashiorkor include: change in skin and hair color (to a rust color) and texture,
fatigue, diarrhea, loss of muscle mass, failure to grow or gain weight, irritability,
enlarged liver (hepatomegaly). What causes the symptoms of kwashiorkor1? For
the answer:
a) specify what proteins everybody needs for nourishing diet:
b) explain the meaning of * nitrogen balance*. Specify the nitrogen balance;
observed in protein starvation and in growing children:
c) enumerate biologically active compounds formed in the human body from
amino acids;
d) point out the pathways which, arc accelerated or slowdown in protein
starvation.
2. The young man with weakness, insomnia, and weight reduction visited a doctor.
During the examination, the doctor found out that young man decided to
be vegetarian and during several last months followed plant diet without any
consultation with the doctor. What arc the possible reasons for the described
.symptoms'? For the answer:
8.2 Transamination ana Deamination of Amino Acids 411

a) give the definition to the * nitrogen balance* and whether or not it is changed
in the patient;
b) point out the cause of changed nitrogen balance;
c) point out compounds do contain, high-value, proteins;
d) specify the metabolic pathways of these compound in the human body:
e) answer the main question of the task.
3. To research different diet influences on human health, volunteers were subjected
to particular experiment. It is known that potatoes practically do not contain
tryptophane and methionine, and the total amount of protein in this vegetable
is 2%. Presume, why prolonged artificial diet containing potatoes and cocktail
includingTrc-Arg-Lys-Pro-lle peptide can induce damage to metabolism in the
human body? For the answer:
a) write down the formula of this peptide;
b) explain, whether this peptide being digested in stomach or not;
c) enumerate peptidases which can take pan in given peptide digestion in small
intestine; name proenzymes of these enzymes, mechanism of activation and
activators;
d) specify the group due to the possibility to be synthesized in the body these
amino acids belong to. Explain whether nitrogen balance is changed or not
in animals on this diet;
c) answer the main question of the task.
4. A weight-reducing diet heavily promoted some years ago required the daily
intake of liquid protein* (soup of hydrolyzed gelatin), water, and an assortment
of vitamins. All other food and drink were to be avoided. People on this diet
typically lost 10 to I4 pounds in the first week. Opponents argued that the weight
loss was almost entirely due to water loss and would be regained very soon after
a normal diet was resumed. What is the biochemical basis for this argument? For
the answer:
a) a number of people on this diet died. Explain some of the dangers inherent in
the diet, and how it can lead to death;
b) enumerate essential components which arc shortened in such diet:
c) present particular schemes to confirm your answer.
5. The inflammation associated with atrophic gastritis and a damage to the
stomach lining causes digestive problems; nutrient deficiencies arc observed as
well. Patients suffer from weight loss, vomiting, lack of appetite, nausea, iron
deficiency anemia, pain in the stomach. Why these symptoms occur in damaged
protein digestion in the stomach? For the answer:
a) point out the value of gastric juice pH in healthy people;
b) specify functions of HCI;
c) describe wrhich peptidase takes part in protein digestion in stomach: peculia­
rity of its activation and specificity.
6. Amino acid alanine containing N15 isotope in a-amino group was administrated
to mouse with food. It was found that the N15has rapidly appeared in a-amino
groups of aspartate, glutamate and other amino acids in the liver (with the
exception of lysine and threonine). Explain, why does it occur? For the answer:
 Chapter 8. Nitrogen metabolism

a) draw the reactions of aspartate and glutamate formation with alanine

participation;
b) describe enzymes catalyzing these types of reactions: specify coenzyme;
c) specify the group of amino acids due to the possibility that they have been
synthesized in the body and Glu, Ala, and Asp relate to.
7. To improve the digestion of food proteins in chronic inflammatory-dystrophic
disorders of the pancreatic gland, stomach or intestine patients arc prescribed
mczym forte (pancreatin) containing pancreatic enzymes. What is the remedial
mechanism of this drug? For the answer:
a) enumerate enzymes digesting proteins in. the gastrointestinal tract;
b) specify the mechanism of proteolytic enzyme acti vation:
c) draw the schemes of these enzymes activation, point out the activators;
d) explain the specificity of proteases;
e) describe the consequences of these enzymes activation in the tissue of
pancreas.
8. I n patients after prolonged hepatitis, the ALT and AST activities were measured
in the blood scrum. What transaminase activity is increased to a greater extent,
and why? For the answer:
a) explain the meaning of the enzyme diagnostics;
b) draw the scheme of reactions catalyzed by ALT and AST;
cl point out coenzyme of these reactions: describe vitamin from which this
coenzyme is derived;
d) describe the biological importance of this type of reactions in amino acid
metabolism;
e) specify the demands which arc claimed to enzymes been used in enzyme
diagnostics.
9. An acute myocardial infarction, a thrombus of a coronary artery and ischemia
results in necrosis. Patients complain of chest and epigastric pain radiating to
the arms, shoulders, neck and associated with sweating, nausea and shortness
of breath. Which scrum enzyme activity measurement is used in clinic for
differential diagnosis (from angina pectoris, acute pancreatitis, perforated
stomach ulcer, etc.)? For the answer:
a) enumerate transaminases which are measured in serum enzyme diagnostics.
Write down reactions catalyzed by these enzymes;
b) specify the coenzyme of these enzymes;
c) explain the importance of these reactions in amino acid metabolism;
d) point out other enzymes and proteins measured in serum enzyme diagnostics
in myocardial infarction.
10. Patient was admitted to the hospital with complaints on malnutrition, light­
colored feces, urine colored as thrown ale*; it was revealed icteric discolor of
skin and sclera. Due to examination and laboratory' serum tests, the doctor
diagnosed viral hepatitis. W'hy blood lest is revealed the considerable increase
of transaminase activity (ALT and AST) in this patient? For the answer.
a) explain the reason for mentioned enzymes increased activity in the blood;
b) specify, what enzymic activity is increased in the largest degree in this
pathology and why;
8.3. Ammonia Detoxification. Urea Cycle, urea Cycle Disorders, Hyperammonemia 413

c) draw the graph indicating the change ofthese enzymes activity in this disorder;
d) write down reactions catalyzed by these enzymes, give full names of direct
and reversible reactions, specify coenzyme;
e) point out the Ritz ratio in this disorder.
II. The most highly sensitive lest in viral hepatitis is y-Glutamyl transpeptidase
(y-GT) increased activity in blood, wrhich level rises 10—15 times more than
the norm (30-50 ME/L). What is the diagnostic value of this enzyme? For the
answer
a) present the main properties of enzymes which activity determination in
patient blood are widely used in. enzyme diagnostics;
b) explain the y-Glutamyl transpeptidase (y-GT) system and present an
appropriate scheme;
c) givc examples of other enzymes which activity is determined in the liver
disorders.

83. AMMONIA DETOXIFICATION. UREA CYCLE. UREA CYCLE

DISORDERS, HYPERAMMONEMIA

Waste products of nitrogen metabolism

Pro Leins are the source ofover 95% of the total amount of excreted nitrogen; nucleic
acids make up the remaining 5%. The major problem is to get rid of the ammonia
(NH.() that forms when amino groups (-NHJ are removed from amino acids during
protein catabolism. A certain amount of ammonia is formed in the degradation of
other nitrogcn-containing compounds (biogenic amines and nucleotides). NH. is
formed in the interline, as well, by the action of microbes (putrefaction) and enter w
portae; its concentration in portal blood is much more than in blood stream, and it is
also detoxificatcd in the liver (Fig. 8. 14).
Ammonia, urea and uric acid (sec below) are the most common nitrogenous waste
products, Era? ts synthesized in the fiver an<7 about of 95% of ammonia is excreted as
urea; ammonia salts formed in the kidney involve 5% ofall excreted nitrogen.
The catabolic prod Lie lion, of ammonia poses a serious biochemical problem
because ammonia is a very toxic molecule, which easily passes through biological
membranes it is kept in low concentrations. In the blood of humans, the range
of ammonia is within 25-40 pM/L, Ammonia is a relatively strong base, and al
physiological pH values the majority of ammonia exists as ammonium ion NH4+
which can’t pass biological membranes;

H H 1+

h :n: + h*■—> h :n:h

H H

The intensity rate of amino acid’s deamination in tissues is high but low ammonia
levels in bloodstream is the result of active binding of ammonia to non-toxic
compounds (glutamine and alanine) transporting it to the liver and kidneys.
414 Chapter 8. Nitrogen metabolism

Biogenic amines Aminoacids Nucleotides

k )

Alanine Glutamate Ammonia salts

-25g/day -0.5g/day

Muscles. Drain Muscles.

Liver Brain Kidney
ana otn&r tissues intestine
!-

Fig 8.14. Summary of ammonia sources and pathways of its metabolism in different tissues

Glutamine synthesis. Glutamine transports ammonia in the bloodstream.

In most animal tissues, much of the free ammonia is converted to a non toxic
compound - glutamine, which is exported from the extrahepatic tissues into the blood
and then to the liver or kidneys.
The free ammonia produced in tissues is combined with glutamate to yield
glutamine by the action of glutamine synthetase, which requires ATP and Mg2' ions:

COO- COO-
I
H—C— NHq ATP ADP + Pi
I
H—C— NHt

I
CH2 + NH:3 < 2. I
ch2 + h2o

(Jh2
Glutamine
synthetase
I
C-0

COO" nh2

Glutamate Glutamine

Glutamine synthetase is a mitochondrial enzyme that is found in a variety of

tissues; the enzyme possesses high affinity to ammonia and due to this reaction low
NHi' concentration is held in tissues and blood. Glutamine synthetase is one of the
main regulatory enzymes in the amino acid metabolism and allosterically inhibited
by AMP, glucose-6-phosphalc, as well as Gly, Ala, and His. Glutamine is the neutral
compound and easily passes through biological membranes. Glutamine is normally
present in the blood in much higher concent rations than other amino acids, in human
body glutamine formed in different tissues travels to the liver, kidneys, and intestine
where glutaminase removes the anti de nitrogen to form glutamate:
8.3. Ammonia Detoxification. Urea Cycle, urea Cycle Disorders, Hyperammonemia 415

COO’ COO"

f J— C
1 NH3
1
H-€“" NHg
I
ch2
+ H^O 1
CHn + NH3
1
c—o
Glutaminase 1
1 CHe

nh2 COO-

Glutamine Gtulamate

nh5+ h -nh4
Glutaminase in the kidneys releases ammonia, which is excreted as ammonia softs
in exchange for one and two valence cations. This mechanism is one of the main of
maintenance of acid-base balance and preservation of important cations lor die body,
yf/nmon/a cxcat/km wf/A urine is ft 5— /. 2 hour
In metabolic acidosis, there is an increase in glutamine processing by the kidneys
due to induced glutaminase activity. Produced ammonia is used to neutralize acidic
products and forms salts with metabolic acids | mainly NH^CL (NH facilitating
their excretion in the urine. .Ammonia salts excretion can reach IO g/24h in acidosis.
This mechanism of ammonia excretion mainmins acid-base balance and ■Vo4’
and X\/br Auman body: the loss of the same amount of Na' could lead to decreasing of
osmotic pressureof interstitial fluid and blood, and this could result in water depletion.
In the intestinal cells, the same enzyme (glutaminase) hydrolytically liberates
amide nitrogen from glutamine:

Glutamine 4- H/) - Glutamate + NH.

Glutaminase

Formed glutamate undergoes transamination with pyruvate, and the last is

converted to alanine. Thus, in entcrocytcs amide group of glutamine is converted
to ammonia, and the amino group of glutamate is included in alanine composition.
Large quantities of alanine pass from the intestine to the blood of portal vein and arc
taken in by the liver (Fig. 8.15).

Ammonia detoxification in the muscle tissue

Alanine plays a special role in transporting amino groups to the liver in a nontoxic
form, via a pathway called the glucose-alamne cycle ( Fig. 8.16). In the muscle and
certain other tissues amino groups arc transferred to pyruvate, a readily available
product of muscle glycolysis, by the action of alanine aminotransferase. The formed
alanine passes into the blood and travels to the liver.
416 Chapter 8. Nitrogen metabolism

TISSUE BLOOD LIVER

Muscle, intestine A. D.

urine
Ammonia salts
I
Urea

Figu &1& Pathways of amino acid nitrogen, and ammonia metabolism. A — elimination of nitrogen
from muscles and intestine in the composition of alanine and giuiamine; B — elimination of nitrogen
from drain and muscles in the form of glutamine; C — ammonia excretion as ammonia salts Dy kidney;
D - inclusion of amino acid nitrogen into urea in the liver; CP — creatine phosphate

In the cytosol of hepatocytes, alanine aminotransferase transfers the amino group

from alanine tocx-kctogluiarate, forming pyruvate and glutamate. Glutamate can then
enter mitochond ria, where the glutamate dehydrogenase reaction releases NH*, or can
undergo transamination, with oxaloacctatc to form aspartate, another nitrogen donor
in urea synthesis, (see below). The use of alanine to transport ammonia from skeletal
muscles to the liver is another example of the intrinsic economy of living organisms.
Vigorously contracting skeletal muscles operate anaerobically; producing pyruvate and
lactate from glycolysis as well as ammonia from protein breakdown.
8.3. Ammonia Detoxification. Urea Cycle, urea Cycle Disorders, Hyperammonemia 417

Liver Muscte
Glucose
Glucose Glucose

2 NAD"
Muscle proteins
2 NADH
Gluconeogenesis
2 NADH
Glycolysis I
Amino acids
BLOOD

2 Pyruvate 2 Pyruvate

Biosymnelw aminolransiefase
pain ways

2 Alanine
2 Alanine 2 Alanine

Fig. 8.16.The giucose/aianine cycle, within ine muscle, amino acid degradation leads to me transfer
of NHj-group to n-ketogrutaraie and pyruvate. Trie formed alanine travels to me liver, wnere the
cartoons of alanine are used for gluconeogenesis and me alanine nitrogen is used for urea synthesis.
This coufd occur during exercise, when the muscle uses blood-borne glucose

These products must find their way to the liver where pyruvate and lactate arc
converted into glucose by gluconeogenesis, which is returned to the muscles, and
a m mo nia i s co nvc tied to urea for exo ret io n. T he g lucosc -a lan i nc cvc le, i n co ncert w it h
the Cori cycle, accomplishes this transaction. The energetic burden ofgluconeogenesis
is thus imposed on the liver rather than the muscle, and all available ATP in muscle is
devoted to muscle contraction.

Ammonia fixation in brain

The reaction of the reduefm? //ic ammafion o/a-toog/utarate under the action of
glutamate JeJiyJrogenasc (which catalyzes the reverse oxidation reaction) may be used
for ammonia detoxification in the brain and! some other organs. With the following
glutamine formation, this rout is beneficial for brain cells because it ensures the
binding of two NH. molecules (Fig. 8.17).

NADPH IMADP* + H<: ATP ADP+ Pj t h.2o

Glutamate Giutamme
defiytfiageiiase synth erase
(GDW) COD’

u-KetoglularaDe L-Glulamale Glutamine

Fig. 8.17. Ammonia fixation in brain and some other tissues

418 Chapter 8. Nitrogen metabolism

Urea cycle (ornithine cycle, Krebs-Henseleit cycle)

In ureotelic organisms, the ammonia deposited in the mitochondria of hepatocytes
is convened to urea in the urea cycle. This pathway was discovered in 1932 by Hans
Krebs (who later also discovered the citric acid cycle) and Kun Hcnsclcit.
Urea production occurs almost exclusively in the liver and is the fate of most of the
ammonia channeled there.
L'nw £r /Ac mai/r waste pmdaef oj nitrogen merabo&m, 90% of al I body nitrogen is
released as urea. Llrca (H.N-CO-NHp is the diamidc of carbonic acid. In contrast
to ammonia, it is highly soluble in water, neutral and therefore relatively non-loxic.
As a small uncharged molecule, urea is able to cross biological membranes easily. In
addition, it is easily transported in the blood and excreted in the urine.

Urea is produced from ammonia in five enzymatic steps

The urea cycle begins inside liver mitochondria, but three of the subsequent steps
take place in the cytosol the cycle thus spans two cellular compartments.
The first amino group to enter the urea cycle is derived from ammonia in. the
mitochondrial matrix ■ ■ NH1 arising by the pathways described above. Some of the
glutamate produced in the gtataminase reaction may be further processed in the liver
by glutamate dehydrogenase, releasing more ammonia and producing carbon skeletons
for metabolic fuel.
Whatever its source, the NHj generated in the liver mitochondria is immediately
used, together with COZ (as HCOp produced by mitochondrial respiration, to form
carbamoyl phosphate in the matrix. This ATP-de pendent reaction is catalyzed by
carbamoyl phosphate synthetase 1 (CPS-t), a regulatory enzyme:

2 ATP 2ADP + Pf

NHJ
J
+ HCOj ------- —-<£.---------- -
I
HjN —C—O—P— o-
I
CPS-I
OH
Cartoamoylphosphate

The carbamoyl phosphate, which functions as an activated carbamoyl group donor,

now enters the urea cycle {Fig. 8.18).
The urea cycle has four enzymatic steps. First, carbamoyl phosphate donates its
carbamoy/ group to ornithine to form ciirarWow, with the release of Pi. Ornithine is
non-protcinogcnic amino acid, that plays a role resembling that of oxaloacctalc in the
citric acid cycle, accepting material al each turn of the cycle. The reaction is catalyzed
by ornithine transcarbamoylase (ornithine carbamoyl transferase), and the citrulline
(which is also non-proteinogenic) passes from the mitochondrion to the cytosol
8.3. Ammonia Detoxification. Urea Cycle, urea Cycle Disorders, Hyperammonemia 419

COO"
I
+ HqN—C—H
I
ocxr (CHa)a
I -Pi |
NH? + -C —H ■------------ ---- ----------- NH
I I Ornithine
transcarbamoylase A q
C—'O iCH2)a
I
OPO2.H2 NH2 NH2

Carbamoyl phosphate Ornitine Gitinline

The second amino group now enters from aspartate (generated in mitochondria by
transamination and transported into the cytosol) by a condensation reaction between the
amino group of aspartate and carbonyl group of citrulline, forming
This cytosolic reaction, catalyzed by argininosuccinate synthetase, requires ATP:

COO" COO"

HaN—C—H %N-—C—H
1
(CH2h COQ- (CH2h COO"
1 ATP AMP-PP; 1
ch2
NH
1
C=iO
11
+ p

’H-jjN— C — H
....... 5 1
.A.
Argjnirtosuccinase
NH
|
C—N — C — H
synthetase 1
nh2 coo nh2 COO

Citrulina Asparate Arpininosuccinate

Thc argminosuccinatc is then cleaved by argininosuccinase (argininosuccinatc

lyase) to form free aigm/nt’ and/urmvraA?, the latter entering mitochondria to join the
pool of citric acid cycle intermediates:

(CHgjg COO“ COO-

NH CHj CH

Argimnosuccinas-e
tyase
COO"

Argininasuccinate Fumarate Arginine

This is the only reversible step in the urea cycle. In the last reaction of the urea
cycle, the cytosolic enzyme arginase cleaves arginine to yield urea and ornithine:
420 Chapter 8. Nitrogen metabolism

COO"

H.N—C—H
I
(CH2h COO" NH2
*h2o I
NH + HN—C —H + C=O
Arginase
I
I I
C = NH (CHzh nh2
I
nh2 NHz

Arginine Ornitine urea

Ornithine is transported into the mitochondrion to initiate another round of the urea
cycle.

COO"

H^hl*C-H
CHa
COCr
l ■ Aspartate

Fig. 8.18 Urea eycle

8.3. Ammonia Detoxification. Urea Cycle, urea Cycle Disorders, Hyperammonemia 421

Genesis of urea atoms: amino groups of urea arc derived from ammonia, formed
by amino acid deamination orcntcring from blood (mainly portal veins), and aspartic
acid:

Ammonia Aspartic acid

Omit hi no cycle consumes 4 maciocrgic bonds of 3 ATP per turn. Anyway, the
process of urea synthesis has possibility to compensate energy inputs (Fig. 8.19):
► when aspartate is regenerated al the stage of malate dehydrogenation (fumarate -*
malate) - NADH formed provides 3 ATP molecules by oxidation in electron
transport chain:
► glutamate oxidative deamination yields NADH and provides 3 ATP, as well.

Fig. 8.19. The Krebs bicycle, indicating the common steps between the TCA and urea cycles

The urea cycle and the citric acid cycle arc independent cycles but arc linked.
Aspartate formed in mitochondria by transamination between oxaloacetate and
422 Chapter 8. Nitrogen metabolism

glutamate can be transported to the cytosol, where it serves as nitrogen donor in

the urea cycle reaction catalyzed by argini no succinate synthetase. These reactions
making up the aspartate-argininosuccinate shunt provide metabolic links between
the separate pathways by which the amino groups and carbon skeletons of amino
acids arc processed.
CO3 is produced in the TCA cycle by isocitrate andu-ketoglutaraic decarboxylation.
ATP for the urea cycle is provided by the Krebs cycle, as well.
Urea cycle in the liver performs two ftinclions:
► the conversion of amino acid nitrogen into urea excreted by kidney helps to
escape accumulation of toxic ammonia in the body;
► arginine synthesis to replenish its pool in the body.

The activity of the urea cyde is regulated at two levels

In general, the urea cycle is regulated by substrate availability; the higher the rate
of ammonia production, the higher the rate of urea formation Regulation by substrate
availability is a general characteristic of disposal pathways, such as the urea cycle,
which remove toxic compounds from the body.
On a shorter time scale, allosteric regulation of al least one key enzyme adjusts the
flux through the urea cycle. The first enzyme in the pathway, carbamoyl phosphate
synthetase I. is allostcrically activated by N-acetylglutamate (Fig. 8.18), which is
synthesized from acetyl-CoA and glutamate by N-acelylglu tarn ate synthase. In
mammals N-acetylglutamate synthase activity in the liver has a purely regulatory
function (mammals lack the other enzymes needed to convert glutamate to arginine).
The steady-stale levels of N-acctylglu tamale arc determined by the concentrations
of glutamate and acetyl-CoA (the substrates for N-acctylglutamale synthase) and
arginine (an activator of N-acetylglutamate synthase, and thus an activator of the urea
cycle) (Fig. 8.20).
Cortex hormone cortisol induces the synthesis of carbamoyl synthetase 1. ornithine
carbamoyl transferase, and arginase.
Those changes in demand for urea cycle activity arc mol over the long term by
regulation of the rates of synthesis of the four urea cycle enzymes and carbamoyl
phosphate synthetase I in the liver. All five enzymes arc synthesized at higher rates
in starving individuals, in prolonged intensive physical work, and in individuals on
very-high-protein diets than in well-fed ones eating primarily carbohydrates and
fats. Individuals on protein-free diets produce lower levels of urea cycle enzymes.
Disorders characterized by intensive protein degradation (diabetes mcllitus, etc.) arc
accompanied by urea cycle enzyme activation, as well.
Urea enters the bloodstream (w/ra b/aod cance/rirafion is 2.5—5.4 mM/Lj and
exerted by kidney in the a mo uni of 25—40 gram/24 hours.
8.3. Ammonia Detoxification. Urea Cycle, urea Cycle Disorders, Hyperammonemia 423

COO"
I
+H.3N—C — H

H—C — H Glutamale
Acefiyi-'CoA
H—C—H

COO-

N-Aceiylgluiamate
sy-ftiJiase Arginine

CoA-SH

O COO"
I I
che c-h

N-AcetyfaJutamate

; I
H —C — H
I
COCF

2 ATP 1 2 ADP + P, O 0

nh4+
1 I
rtCO3+ + HgN---- C---- O---- P----- O’
Carbamoyl phosphate
syflihasc l cr
Carbamoyl phosphate

Fig. 8.20. Activation of carttamoyl phosphate synthetase L Argmine stimulates me synmesis of

N-acetylgiutamate, which activates CPS!

Ammonia is toxic to animals

Asammonia is converted to urea in the liver, increased blood ammonia concentration
(hyperammonemia I is observed in liver disorders (hepatitis, cirrhosis, etc.). or inherited
defects of urea cycle enzymes (see later). Clinical symptoms of hyperammonemia involve
nausea, vomiting, excitement, irritation, sleepiness, dizzy spells, and cramps, break of
coordination, cerebral edema (an increase in the brain's water content) and increased
cranial pressure, unconsciousness, coma, and death.
The mechanisms of ammonia toxicity.
I. Ridding the cytosol of excess ammonia requires reductive amination of
a-ketogIutarale to glutamate by glutamate dehydrogenase and conversion of
glutamate to glutamine by glutamine synthetase. Both enzymes arc present at
high levels in the brain, although the glutamine synthetase reaction is almost
certainly the more important pathway for removal of ammonia. Leakage of
a-kefog/uftirafe fefock-t the 7 CA cyc/er/itif teaJ fo hypoenc^erfc sfa/e and decreasing
oppression of amino acid metabolism (transamination).
424 Chapter 8. Nitrogen metabolism

2. High levels of NH4' lead to increased levels of glutamine, which acts as an

ac/m? solute (osmolytc) in brain astrocytes, star-shaped cells of the
nervous system that provide nutrients, support, and insulation for neurons. This
triggers uptake of water into the astrocytes to maintain osmotic balance. leadwg
toswe/frrrgand the symptoms noted above.
3. Depletion of glutamate in the glutamine synthetase reaction may have additional
effects on the brain. GYuiamaie and its derivative /GzlB/f) are
important neurotransmitters: the sensitivity of the brain to ammonia may reflect
a depletion ofneuirtransmittera as well as changes in cellular osmotic balance.
This leads to the increase of neuro-muscular excitement and causes convulsions.
4. Blood pH is displaced to cZLj/osls Lhat increases the affinity of Hb to oxygen and
results in tissue and brain /rypoxfar.
5. Abundance ofN H] inwis /raw-s/jar/ of Na ’ and K and competes
with these ions for ionic channels. This also influences and disturbs the
conduction of nerve impulses.

Genetic defects in the urea cyde can be life-threatening

People with genetic defects in any enzyme involved in urea formation cannot
tolerate protein-rich diets (Table 8.2). Amino acids ingested in excess of the mw/w/
daily requirements for protein synthesis arc deaminated in the liver, producing free
ammonia that cannot he converted to urea and exported into the bloodstream, and,
as was noted above, ammonia is highly toxic. The absence of a urea cycle enzyme can
result in hyperammonemia or in the build-up of one ar more urea cycle mtermedia/es,
depending on the enzyme that is missing. Given that most urea cycle steps arc
irreversible, the absent enzyme activity can often be identified by determining which
e>!cZe intermediate fspreseuf m especially elevatedeoncentmfion in die bloodand/or urine.
Table 82. Hereditary enzyme defects of tile urea cycle

N-Acelyiglutamate Hyperammonemia that may be Lethargy: persistent vomiting; poor

synthase accompanied by high plasma feeding: hyperventilation; enlarged liver,
concentrations ot alanine and seizures
glutamine

Carbamoyl phosphate Hyperammonemia; tow citrulline: Lethargy: coma; seizures; vomiting:

synthetase respiratory alkalosis poor feeding; hyperventilation;
hepatomegaly

Ornithine Hyperammonemia: respiratory Seizures; vomiting: poor lee-ding;

tianscarbamylase alcalosis; elevated orotic acid in urine hyperve nti latio n; h e patomega ly

Argi nosuccinate Citrulline mia Lethargy: coma: seizures; vomiting:

synthetase poor feeding; hepatomegaly

Argi nosucci nate lyase Elevated arginosuccinic acid in urine Lethargy; seizures: vomiting; poor
feeding: hyperventilation; hepatomegaly

Arginase Markedly elevated plasma arginine, Delayed development; protein

lactate, and CSF glutamine, and intolerance; spasticity; loss of muscle
modestly elevated blood ammonia control; seizures; irritability
CSr — CHtivospinai fluid
8.3. Ammonia Detoxification. Urea Cycle, urea Cycle Disorders, Hyperammonemia 425

Pnmtfry ftj/wrammcNM'jw/a is caused by several inborn errors of metabolism that

arc characterized by reduced activity of any of the enzymes in the urea cycle. Enzyme
deficiency leads to the accumulation of the substrate of blocked reaction and reduction
of the product; each metabolic block is characterized by elevated levels of ammonia
in die blood.
Hyperammonemia Type 1. A consequence of ctirftcrmoy/ phosphate synthase /
deficiency. this relatively infrequent condition probably is familial.
Hyperammonemia Type 2. A deficiency of omtZhmc transcarhamoy/ase produces
this X chromosome-1 inked deficiency. The mothers also exhibit hyperammonemia
and an aversion to high-protein foods. Levels of glutamine arc elevated in blood,
cerebrospinal fluid, and urine, probably due to enhanced glutamine synthesis in
response to elevated levels of tissue ammonia.
Citnilliniemia. Patients lack detectable (?^f7n/?o5wccj>k7/c syjrfAasf activity. In this
rare disorder plasma and cerebrospinal 11uid citrulline levels arc elevated and I -2 g of
citrulline are excreted daily. Citrulline and argininosuccinate, which contain nitrogen
destined for urea synthesis, serve as alternative carriers of excess nitrogen.
Argininosuccinic aciduria. The metabol ic defect is the absence ofd^7nfflojwcc/7Msc.
A rare disease characterized by elevated levels of argininosuccinatc in blood,
cerebrospinal fluid, and urine is associated with friable, tufted hair (trichorrhexis
nodosa). As for citrullincmia, feeding arginine and benzoate promotes nitrogen
excretion.
Hyperargin inemia. This defect is characterized by elevated blood and cerebrospinal
fluid arginine levels.
Acquired hyperammonemia (secondary) is much more frequent and usually
caused by diseases that result in cither acute liver failure, such as overwhelming
hepatitis B or exposure to hepatotoxins, or cirrhosis of the liver with chronic liver
failure. Chronic hepatitis B. chronic hepatitis C, and excessive alcohol consumption
are common causes of cwTAtwis. The physiologic consequences of cirrhosis include
shunting of blood from the liver to the inferior vena cava, resulting in decreased
filtration of blood and removal of nitrogen-con tai ning toxins by the liver that result
in hyperammonemia.
To reduce blood ammonia concentration and relieve patient condition several ways
arc used: first of all, it is the d ietary restriction of proteins. Although the breakdown
of amino acids can have serious health consequences in individuals with urea cycle
deficiencies, a protein-free diet is not a treatment option. Humans arc incapable of
synthesizing half of the 20 common amino acids, and these essential amino acids
must be provided in the diet. The next way is the introduction of ornithine cycle
intermediates (arginine, citrulline, and glutamate). For example, arginine feeding
enhances excretion of citrulline in patients (stimulation of ammonia excretion
stimulates bypassed ammonia eliminationk Similarly, feeding benzoate diverts
ammonia nitrogen to hippurate via glycine (benzoic acid conjugates with Gly forms
hippuric acid which readily excreted in the urine; and Gly can be synthesized from
CO, and NH,in quantities needed for endogenous protein synthesis). Phcnyiacctic
acid" conjugates with Gin forming phenylacctylglutaminc which is readily excreted in
the urine as well (Fig. 8.21).
426 Chapter 8. Nitrogen metabolism

Glycine

Benzoic acid Hippuric acid

Glutamine

Fig. 8.21. Synthesis of hi pp uric acid and pn^enylacetylgl ulaiTiine

The quantitative characteristics of the metabolism of amino acids leant! in this section
Ammonia concentration in blood sennit 0.04-0.07 mg/OL {25-40 pmol/L)
Urea blood serum concentration; 15-50 rng/dt (2.5-84 mMol/L)
Urea excretion: 25-40 g/24b
Ammonia sails excretion: 0.5-15 g/24h

Review tests
1. The following compounds lake part tn I he reaction:

COO co—nh2
H-—y—H

H — C— H

H— C— NH?
I
coo" (2 COO"

a
Components:
A. Glulaminc.
B. ATP.
C. Glutamate.
D. Glu La mine synthetase.
E. NH3
8.3. Ammonia Detoxification. Urea Cycle, urea Cycle Disorders, Hyperammonemia 427

2. The following com pounds lake pari in the reaction:

CO—'NHz COOH
H—i— H
HgO g)
H—H
I o.
H—C—H H—C—H

H—C—NHj H—C — NHg

I
COO" COO"

Compounds:
A. Glutamate.
B. H2O.
C. Glutamine.
D. Glutaminase.
E. NHr
3. Ammonia sources in the human body:

CD ® ®

Processes:
A. Protein putrefaction.
B. Glucose metabolism.
C. Nucleotide metabolism.
D. Amino acid deamination.
E. Urea catabolism.
4. In tissues ammonia is fixed with the formation of:

Compounds:
A. Glutamine.
B. Alanine.
C. Urea.
D. .Ammonia salts.
E. Glutamate.
428 Chapter 8. Nitrogen metabolism

5. Ammonia introduction into these compounds occurs in:

Tissues:
A. Kidney.
B. Liver.
C. Brain.
D. Muscles, intestine.
E. Brain, muscles and other tissues.

H—c — H—C—H

H—C— NHn
r
C h H—C=NHn
I I I
COO" COO" COO"
8.3. Ammonia Detoxification. Urea Cycle, urea Cycle Disorders, Hyperammonemia 429

Enzymes:
A. Arginine succinate lyase.
B. Arginine succinate synthetase.
C. Carbamoyl phosphate synthetase.
D. Arginase.
E. Ornithine carbamoyl transferase.

Situational problems
1. Normal human blood plasma contains all the amino acids required for the
synthesis of body proteins, but not in equal concentrations. Alanine and
glutamine content is much higher than any other amino acid. Suggest, why?
Confirm your answer with the appropriate schemes.
2. In a study, cats were fasted overnight then given a single meal complete in all
amino acids except arginine. Within 2 hours, blood ammonia levels increased
from a normal level of 94 pmol/L Lo 470 pmol/L, and the cals showed
the c Li nical sy m ptorn s of am mo n ia toxic i ty. A coni rol g rou p fed a co mp I etc amino
acid diet., or an amino acid diet, in which arginine was replaced by ornithine
showed no unusual clinical symptoms. What caused the ammonia levels to rise
in the experimental group? Is arginine an essential amino acid in cals? Why or
why not? For the answer:
a) point out the role of fasting in the experiment:
b) explain, why the absence of arginine can lead to ammonia toxicity:
c) explain, the possibility of arginine substitution by ornithine:
d) present the particular schemes to confirm vour answer.
3. A 15-year-old boy presented with a history of episodic vomiting associated
with subacute encephalopathy from the age of one year. There were 2-3 months
of asymptomatic periods between the episodes. He was born of non-
consanguincous parents at lull term had a normal perinatal period with no
evidence ofbirth asphyxia. During the hospital stay he had 2 episodes ofconvulsive
seizures. Investigations revealed high blood ammonia (288 pg/dL, normal
level is 40-80 pg/dL), markedly elevated levels of citrulline (2200 p moles/L,
normal is 1-55 pmolcs/L), and low blood urea (5 mg/dL, normal is around 7 to
20 mg/dL). He was treated with ant iconvulsivcs and sodium benzoate, advised
a restricted protein diet. What is the reason for the presented symptoms? Why
blood urea concentration is reduced? For the answer:
a) specify the compound 90% of body nitrogen is excreted in;
b) write down the process damaged in this patient:
c) point out the enzyme deficient in this clinical case:
d) explain the mechanisms of ammonia toxicity;
e) explain the expediency of prescribed diet and sodium benzoate.
4. Patient came down with the flu and suffered from dizziness, nausea, convulsive
attacks. Ammonia blood concentration is I. mg/dl. In consideration of influenza,
virus influence on the liver, what mechanisms of pathological symptoms
development? For the answer:
a) specify normal ranges of the blood ammonia level;
b) write down damaged metabolic process a nd point out the site of £ he metabolic
block in flu;
430 Chapter 8. Nitrogen metabolism

c) list compounds which would be accumulated in patient's blood:

d) describe mechanisms of ammonia toxicity;
e) point oul cells most suffering from ammonia toxicity.
5. I n the examination ofdiabetes mellitus patient the elevated ketone bodies level till
300 mg/dl was revealed. Why was this accompanied by considerable increasing
ammonia salts excretion till 10 g/day (0,5 g/day in norm)? For the answer;
a Hist pathways of ammonia detoxification in different tissues (brain, muscles,
and liver), present appropriate reactions;
b) specify amino acid which transports ammonia from muscles to kidney,
present the reaction of its synthesis, point out enzyme:
c) give the scheme of process accelerating in acidosis in the kidney, specify its
importance, point out enzyme the synthesis of which is induced under these
circumstances;
d) write down the formulae of salts in the composition of which, ammonia is
excreted in ketonemia.
6. In patient with severe viral hepatitis (80% of liver parenchyma cells were
damaged) decreased blood scrum urea concentration was reveled up to
1.4 mmol/l (2.5-8.4 mniol/1 in the physiological norm). 16 g/day of urea was
excreted (25g/day in the norm). Why was urea content decreased in blood and
urine? For the answer:
a? write down the scheme of the main waste product of nitrogen the metabolism
synthesis, point out its localization in the body and name;
b) specify which substances concentration may increase in the blood of such
patients;
c) specify symptoms accompanied these substances increased concentration
in blood, explain the mechanism of toxicity of one of them;
d) presume recommendations to reduce the manifestation of mentioned
symptoms.
7. A 22-year-old patient suffering argininosuccinic aciduria from little was
prescribed the treatment by administration of kcto-analogs of valine, leucine,
isoleucinc and phenylalanine against the background of reduced protein in the
diet during two weeks. The doctor noted the decrease ofammonia concentration
in plasma front 0.09 mg/dL to 0.04 mg/dL as a result, and arginine succinate
excretion was reduced from 2 to 0.8 g/24h. What is the molecular mechanism of
the curative effect of ami no acids kcto-analogs? For the answer:
a) write down the scheme ofammonia detoxification in the liver:
b) explain the cause of argininosuccinic aciduria;
c) make up the scheme of present amino acid kcto-analogs usage for the
reduction of hypcram monicmia.
8. In patients with Albright-Butler syndrome (kidney acidosis) secretion of H*
into distal tubule lumen is damaged. It is noted polyuria, increased excretion
of sodium and potassium (that leads to hyponatremia and hypokalemia),
hypocalcemia and osteoporosis, and decreased ammonia salt excretion, as well.
What are the biochemical mechanisms of present symptoms development? For
the answer:
a) specify the enzyme the amount and activity of which is increased in kidney in.
acidosis under physiological norm;
8.4. Synthesis of Nmessentiai Amino Acids 431

b) write down the reaction catalyzed by th is enzyme and explain its physiological
significance;
c) point out the reasons for acidosis development and increased loss of sodium
and potassium in given disorder:
9. it is noted recurrent vomiting, weakness, sleepiness, and convulsive attacks, as
well, in patients with an. inherited disorder of ornithine cycle after food intake.
What are the molecular reasons for observable symptoms? For the answer:
a) write down the scheme of process dam aged in these patients, specify enzymes,
defect of which can cause such disturbances of metabolism;
b) explain the biological function of this process;
c) enumerate substances the content of which is increased! in the blood of these
patients;
d) describe the lox icily of one of them on the nervous system.

8A SYNTHESIS OF NONESSENTIAL AMINO ACIDS

The carbon skeleton of the most noncsscntial amino acids (Ala, Asp, Asn, Ser, Gly,
Pro. GIir Gin), and Cys can be synthesized from glucose catabolism intermediates
(Fig. 8.22). a-Ami.no groups are inserted into corresponding a-keto acids by
transamination. Glutamate is the universal amino-group donor.
Several amino acids are synthesized by direct transamination of metabolites of
common catabolic pathways with glutamate:
Gtu a-Ketoglutarate

Alanine: Pyruvate
ALT (PLP)

Gtu ci-Ketoglutarate

Aspartate: Oxaloacetate ■ ■ Aspartate

AST (PUP)

Amino acid Keto acid

k /
Glutamate: a-Ketoglutarate --------- Glutamate
AjTinotransferase (PLP)

► asparagine is formed from aspartate by a reaction in which glutamine provides

the nitrogen for formation of amide group:

ATP AMP+PPj

HgN+

Glutamine Glutamate

0 NH2

Aspartate Asparagine
432 Chapter 8. Nitrogen metabolism

► glutamine is formed from glutamate by glutamine synthetase, which adds N H 3 to

the carboxyl group of the side chain, forming an amide:

NH,
P

COO’ ATP ADP, hfcPO* C=O

1
ch£
f Glutanifte-
CHs NHZ
CH?
f
CH — NHi CH—nh;
i
COO" COO"
Glutamate Glutamine

Fig. 8.22. The pathways of nonessential amino aciEfs biosynthesis

► serine is synthesized from 3-phosphoglycerate, an intermediate of glycolysis

(Ch. 8.6);
► glycine is formed from serine by serine hydroxymethyl transferase with
tetrahydrofolate as a coenzyme (binds serine P-carbon) (Ch. 8.6):

Serine + H4-folalc - Glycine 4- 5.10- Methylene- H ,-folatc + H2O.

8.5. The Main Steps of Amino Acid Degradation; Glucogenic, Kerogenic and Bain Gluco-... 433

Proline is synthesized from glutamate:

Glutamate ------- Glutamate scmialdehyde------------ *■ Proline.

The synthesis of partially essential amino acids Arg and His docs not respond to the
body needs, especially in growth. Some amount of arginine is formed in the ornithine
cycle. Histidine can be synthesized from ribose and ATP.
Conditionally essential amino acids Tyr and Cys arc formed from the essential:
phenylalanine is converted to tyrosine under the action of phenylalanine hydroxylase.
Methionine is the donor of sulfur required for cysteine; serine provides the carbon
skeleton and a-amino group, as well (Ch. 8.6).

8.5. THE MAIN STEPS OF AMINO ACID DEGRADATION;

GLUCOGENIC, KETOGENIC AND BOTH GLUCO AND
KETOGENIC AMINO ACIDS
The carbon skeletons that arc left over after deamination undergo further
degradation in various ways {Fig. 8.23). The 20 protcinogcnic amino acids produce
six different degradation products which are metabolites of common catabolic pathways:
► pyruvate;
► acetyl-Co A;
i a-kctoglutaraic;
► succinyl-Co A;
► fumarate;
» oxaloacetatc.
Amino acids catabolized to pyruvate or other intermediates of TCA
(o-ketoglutarate, succinyl-CoA. fumarate) arc converted to oxaloacetatc and,
therefore, precursors for gluconeogenesis. These amino acids arc referred to as
glucogenic amino acids (aspartate, alanine, glutamate, serine etc.). Cortisol stimulates
glucose synthesis from amino acids, thus inducing the synthesis of enzymes of
gluconeogenesis, ornithine cycle and ALT in the liver.
Amino acids that supply acetyl-CoA or acetoacetate arc, therefore, known as
ketogenic amino acids. Only leucine and lysine arc purely ketogen ic. Several amino
acids yield degradation produces that arc both glucogenic and ketogen ic. This group
includes phenylalanine-, tyrosine, tryptophan, threonine, and isoleu cine.
Amino acid catabolism normally accounts for only 10% to 15% of the human
body’s energy production; the main pathway of amino acid usage is their recruit mem
into the gluconeogenesis that is increased in starvation and diabetes mcllitus.
Nitrogen free residues of amino acids have an anaplerotic effect — i.e., they
replenish the tricarboxylic acid cycle in order to feed the anabolic reactions that
originate in it:

colX
a) Amino acids Pyruvate Oxaloacetatc
434 Chapter 8. Nitrogen metabolism

Enzyme pyruvate carboxylase (coenzyme — biotin) is found in the liver and muscles,

b) G1 u tamate » a- Kctog iuta rate

The conversion occurs in many tissues and is catalyzed by glutamate dehydrogenase,

or aminotransferases. Both these reactions arc the main anaplcrotic.

Valine

c) Propionyl-CoA------- Succinyl-CoA

I so leucine

These reactions occur in many tissues where pyruvate carboxylase is not found.

Amino acids------- ► Fumarate

di
Amino acids Oxafoacciatc

The East two reactions occur in the liver.

Ftr 823. Pathways of nitrogen-free residue ot amino acids usage

8.6. Special Pathways of Some Amino Acids: Phenylalanine MetaDoiism, Tetranytfrofolate... 435

8.6. SPECIAL PATHWAYS OF SOME AMINO ACIDS: PHENYLALANINE

METABOLISM, TETRAHYDROFOLATE, VITAMIN Bn
AND S-ADENOSYLHETHIONINE
Besides metabolic pathways characteristic for the majority of protcinogcnic ami no
acids, there are specific ones leading to the synthesis of biologically important products
governing physiological state of the human body.

Tyrosine and phenylalanine metabolism

Phenylalanine is an essential amino acid: tyrosine is the semi-essential and
synthesized from phenylalanine in the human body (Fig. 8.24). Dietary’ proteins
contain enough, both amino acids.
Phenylalanine is mainly used in two ways: tissue protein synthesis and conversion to
tyrosine that is important to remove the surplus of phenylalanine that is toxic for cells.
Melanin
epinephrine
norepinephrine
dopamine
DOPA

CHj

+HqN—-C—COO
H CO2

L-Phenyiaianins NADP* naDPh + H* L-Tyrosine

Fig. 8.24. Tyrosine syiittiesiSu DHPR — di hydroDi opterin reductase

Phenylalanine hydroxylase (also called phenylalanine-4-monooxygenase) is

one of a general class of enzymes called mixed-function oxidases, which catalyze
simultaneous hydroxylation of a substrate by an oxygen atom of ()3 and reduction
of the other oxygen atom to H,O. Phenylalanine hydroxylase requires the cofactor
tetrahydrobioplerin (H4-biopterin), which carries electrons from NADPH to O, and
becomes oxidized todihydrobioptcrin in the process. It is subsequently reduced by the
enzyme dihydrobiopterin reductase in a reaction that requires NADPH.

Tyrosine metabolism
Tyrosine is used lor protein synthesis as other amino acids: some enzymes involve
tyrosine in their active site (for example, insulin receptor, which is (p/ww pro/em
h'nase); tyrosine is very important as the source for a variety of important metabolites,
as well.
436 Chapter 8. Nitrogen metabolism

In adrenal medulla, brain, and some sympathetic nerve fibers tyrosine isconvertcd
io catechol a mines - various naturally occurring amines: dopamine, epinephrine
(adrenaline), and norepinephrine (noradrenaline) that function as neurotransmitters
and hormones within the human body (Fig. 8.25). Tyrosine hydroxylase (specific
monooxygenase) catalyzes the conversion of tyrosine to DOPA; the reaction
requires tetrahydrobiopterin, O2and Fc1' (the process is analogical to phenylalanine
hydroxylation: see above Fig. 8.24). Dopamine is produced by DO PA de carboxylation
(pyridoxal phosphate is the coenzyme). The following reactions yield
norepinephrine (hydroxylation) and epinephrine (methylation). The particular
catecholamine that is synthesized by a nerve cell, or neuron, depends on which
enzymes are present in that cell. Dopamine, a neurotransmitter widely distributed

CHa—CH—COO-

NHg
[Tyrosine]
Tyrosine hydroxylase
{totranydrofciopterin, Fe2\ O?)

CH2— CH—£>OCr

NHa
DOPA
DOPA decarboxylase
- CO2
[pyridoxaj-pnospnata)

CH2— CHj— NHj

Dopamine
Dopamine Hydroxylase
it h srariy drobiOLitorin;
ascorbate, Qu , O?)

— CHz—NHa

Noradrenaline
Methyltransferase
(S^adenosylmethionine)

Adrenaline

Fig. 8.25. Catecholamines are derived from the amino acid tyrosine
8.6. Special Pathways of Some Amino Acids: Phenylalanine MetaDoiism, Tetranyttrofolate... 437

in the central nervous systemT including the basal ganglia of the brain that collectively
control muscle tone, inhibit movement, and control ircmour. Norepinephrine is a
neurotransmitter of sympathetic nervous system released in response to stress. The
general function of norepinephrine is to mobilize the brain and body for action.
Norepinephrine is important for attentiveness, emotions, sleeping, dreaming, and
learning. Epinephrine (adrenaline) is the hormone of intensive physical activity and
stress; it regulates basal metabolic rate and strengthens cardiac muscle contraction,
as well.
In thyroid gland, tyrosine is the precursor of iodothyronines (thyroxin and
triiodothyronine}, these hormones arc iodinated tyrosine residues.
In melanocytes (skin pigment cells, hair follicles and retina) tyrosine is the precursor
of melanin — high molecular weight hydrophobic pigment (Fig. 8.26). The synthesis
of melanin is a niultistep process. The first two steps in the synthesis of melanin arc
catalyzed by tyrosinase (DOPA oxidase), a fopper-co/j/amiXgoxidase, which converts
DOPA todopaquinonc.

Tyrosine
Tyrosine Hydroxylase .

Tyrosinase D0PA

(02; Oi*]
(DOPA oxidase} l

z DOPA quinone

Emnelanin Eurnemnin Pneomelanin

(mack form) (brown form) {yellow to red)

Fig. 8.26. Tyrosine is me precursor of meraniiiSu DOPA quinone is converted to further products,
eventualFy forming eumelanin and pneomelanin — pigments of Dfack-Drown and yeltow-red color

Degradation of phenylalanine and tyrosine takes place in the liver (Fig. 8.27).
Homogentisic acid is an. intermediary product of tyrosine catabolism and converted
to acid with the cleavage of the aromatic ring. Homogentisate
dioxygenase requires vitamin C and Fe2' (or heme, sometimes Cu2') for catalysis. The
last step in the pathway produces both/wmera/c (which can be used for gluconeogenesis)
and the ketone body ace/Mce/a/c.
438 Chapter 8. Nitrogen metabolism

CH

u-KetogJula/ate Glutamate - CO2

+ Os

Aminotransferase Dioxygenase
CH—NHg

GOO­ coo-
I
Tyrosine P-Hydroxypnenylpyruvato H— C— H

C-0

COO" CHa
H-A—H
Acetoacetate
+ Qa I + h2c
OH C»0
Homogentisate
dioxygenase
I +
H—C —H
(n+C;Fe/+)
I
CH2-COO" C=0 COOT
T

OH
I
CH

CH
CH

Sh
Homogantisic acid I
cocr COO'

Fumarytacetoacetate Fumarate

Fig. 827. Phenylalanine and tyrosine degradation Note, that phenylalanine is not present in tne
scheme (Phe is oxidized to Tyr — see above)

Inborn errors of phenylalanine and tyrosine metabolism

In individuals with PKU (phenylketonuria: inherited defect of phenylalanine
hydroxylase), a secondary, normally little-used pathway ofphenylalanine metabolism
comes into play. In. this pathway, phenylalanine undergoes transamination with,
pyruvate to yield phenyl pyruvate. Phenylalanine and pheny [pyruvate accumulate
in the blood and tissues and arc excreted in. the urine — hence the name
phenylketonuria*:
8.6. Special Pathways of Some Amino Acids: Phenylalanine Metauoiism, Tetrafiyttrofolate... 439

DihydrotHCiplerin Oxidated
Protein synlresrs
Tyraane CaSocfiolamines
Pneflylalartine Thyronne
Hydrcnyla&e Melanin

NADU + H

Fhenyllaclic sod
Hwtuciion

Much of the rather than being excreted as such* is cither

decarboxylated to p/ieny/acetate or reduced to pheffyliactate. Phcnylacctatc imparts a
characteristic odor to the urine (*mousy* odor), which nurses have traditionally used
to detect PKU in infants. The accumulation of phenylalanine or its metabolites in
early life impairs normal development of the brai n, causing severe mental and growth
retardation. This may be caused by excess phenylalanine competing with other amino
acids for transport across the blood-brain barrier, resulting in a deficit of required
metabolites.
The moderate form of PKU is due to inherited deficiency of dihydrobioptcrinc
reductase.
Phenylketonuria was among the first inheritable metabolic defects discovered
in humans. Wien this condition is recognized early in infancy, mental retardation
can largely be prevented by rigid dietary control. The diet must supply only enough
phenylalanine and tyrosine to meet the needs for protein synthesis. Consumption of
protein-rich foods must be curtailed. Natural proteins, such as casein of milk, must
first be hydrolyzed, and much of the phenylalanine removed to provide an appropriate
diet, at least through childhood. Because the artificial sweetener aspartame is a
di peptide of aspanate and the methyl ester of phenylalanine, foods sweetened with
aspartame bear warnings addressed to individuals on phenylalanine-con trolled diets.
In the deficiency of dihydrobiopterin reductase (BH is necessary for proper
activity of the enzyme PAH) — this coenzyme can be supplemented as treatment.
440 Chapter 8. Nitrogen metabolism

Those who suffer from this form of hyperphenylalaninemia may have a deficiency of
tyrosine (which is created from phenylalanine by PAH), in which case treatment is
supple mental ion of tyrosine to account for this deficiency.
Levels of dopamine can be used to distinguish between these two types.
Teirahydrobioptcrin is required to convert Phe to Tyr and is required to convert Tyr
to L-DOPA via the enzyme tyrosine hydroxylase. L-DOPA, in turn, is converted
io dopamine. Low levels of dopamine lead to high levels of prolactin. By contrast,
in classical PKU (without dihydrobioptcrin involvement), prolactin levels would be
relatively normal.
Screening newborns for genetic diseases can be highly cost-effective, especially in
the case of PKU. The tests (no longer relying on urine odor) arc relatively inexpensive,
and the detection and early treatment of PKU in infants (eight to ten cases per
100,000 newborns) saves millions of dollars in later health care costs each year. More
importantly, the emotional trauma avoided by early detection with these simple tests
is inestimable.
Albinism includes a spectrum of clinical symptoms characterized by hypomela nosis
due to heritable defects in eye and skin melanocytes (tyrosine hydroxylase converting
DOPA io dopaquinone is affected: Fig. 8.28). Hair bulbs from these patients fail to
convert added tyrosine to pigment melanin and melanocytes contain unpig me med
mclanosomes. Patients suffer decreased visual acuity and photophobia. Prolonged
insolation can lead to burnsand skin cancer.
Another inheritable disease of phenylalanine and tyrosine catabolism is
alkaptonuria, in which the defective enzyme is homogentisate dioxygenase (Fig. 8.28).
Less serious than PKU. this condition produces few ill effects, although large amounts
of homogentisate arc excreted and its oxidation turns the urine black. Accumulation
of homogentisic acid can. occur in cartilage, casing arthritis. There’s no specific
treatment for alkaptonuria. Treatments arc focused on preventing and relieving
possible complications: arthritis, heart disease, and kidney stones.
Phenylalanine
BH I Pheny
i hydroxylase
Homogentisic acid Tyrosine

Tyrosine
* hydroxylase

Fumarylacetoacetate DOPA ---------- Melanin

Norepinephrine
SAMe

Epinephrine

Fig. 8.28. inborn errors of phenylalanine and tyrosine metabolism

8.6. Special Pathways of Some Amino Acids: Phenylalanine MetaDoiism, Tetranytfrofolate... 441

The neurological disorder Parkinson's disease is associated with an

underproduction of dopamine in substantia nigra and the most spared neurological
disorder (the occurrence is 1:200 among people upwards of 60 years). Parkinson's
disease is caused by reduced activity of tyrosine hydroxylase or DOPA decarboxylase.
Symptoms involve akinesia (constraint movement)* muscular rigidity, and tremor.
Dopamine can't penetrate hematoencephalic barrier and Parkinson’s disease has
traditionally been treated by administering L-DOPa and DO Pa-derivatives (levodopa,
madopar etc.), or MAO-inhibitors to prevent DOPaminc inactivation (nialamide,
pirasidol etc.).
Norepinephrine plays a role in mood disorders such as manic depression which arc
often linked with reduced content of both norepinephrine and DOPaminc in nerve
endings. Overproduction of dopamine in the brain may cause psychological disorders
such as schizophrenia.

Serine and glycine metabolism. Folic acid function

Serine is a non-essential amino acid synthesized from 3-phosphoglyceratc,
an intermediate of glycolysis (E( — 3-phosphoglyccratc dehydrogenase: Ez —
transaminase, coenzyme PLP: E- — 3-phosphoscrinc phosphatase):

COO" NAD COO" COO" COO"

I. NADH Gin KG
-Z VZ -©
H—C<—OH G=-O HqN— C — H -
I E-1 | Eg Eg
CHZ Cl-h GHs ch2
I 1
0 o-0 0—0 OH

3-Phospnogiycerate 3-PnospnonydrQxypyruvate a-Pno&progiycerate Serine

The major sites of serine synthesis arc the liver and kidney.
1 n human body, serine is involved into protein synthesis r first of all, and is found in
many enzyme active sites, as well (serine proteases, for example). Serine can be used
for synthesis of sphingosine-containing lipids, phosphatidylscrinc, glycerol, cysteine,
and glucose. Serine is degraded lo form pyruvate (sec above).
Glycine is a non-essential amino acid: serine (three carbons) is the precursor of
glycine (two carbons) through removal of a carbon atom by serine hydroxymethyl
transferase:
442 Chapter 8. Nitrogen metabolism

nh5

HO
L-Serine
!
Serine hydraxymeUiyl
Iransferase (Pl_P]

M^nyiens-Hd-foiaie

Glycine

The coenzyme in the reaction is tetrahydrofolate (TH I7. H4-folate) which binds
P-carb on of serine forming methylene-11^-folate. The reaction is reversible and glycine
is converted to serine by enzymatic addition of a hydroxymethyl group that requires
the coenzymes methylene-tetrahydrofolate and pyridoxal phosphate.
Glycine is abundant in. proteins, for example, collagen involves 33% of this amino
acid. Glycine is the precursor of glutathione, creatine, heme, purine nucleotides,
coenzymes etc.
Glycine undergoes the cleavage to COr NH‘, and a methylene group (-CH,-).
This readily reversible reaction, catalyzed by glycine cleavage enzyme (called glycine
synthase. as well), requires tetra hydrofol ate, which accepts the meth dene group. 1 n this
oxidative cleavage pathway the two carbon, atoms of glycine do not enter the citric
acid cycle. One carbon is lost as CO and the other becomes the methylene group of
A/5, Ar"-methyl one-tetrahydrofo late, a one-carbon group donor in certain biosynthetic
pathways:
NHq
I
ch2xc^o-

I
0
Glydne

H*-folate

Glycine
synthase
NE,.N10-CH2-H4-fulate
(mettiylena-HrloJate)
CO2+NHj+NADH+ Hh
8.6. Special Pathways of Some Amino Acids: Phenylalanine MetaDoiism, Tetrafiytf rofoiate... 443

Folic acid (vitamin Be or vitamin Bg)

This vitamin is widely spread in nature and found in veast, meat, kidney, liver, leafy
vegetables, as well as, produced by intestinal micro flora. Daily re<ywiremenf5<//o/ic acid
are 50- 200 pg (fill 400 pg) per day.
Folic acid is reduced to H4-folate (tetrahyd no folate) in the liver cells by /Wwctases
which require NADPH as a donor of reducing equivalents (Fig. 8.29).

Folate + NADPH+ H’ ---------- -------------------------- * 7,8 Dihytirofolate + NADP+

Folate reductase

7,8 DinydfOrOiato + naDPh + Hr - - - ------- —► Tetranydrofoiate + naDP+

Dihydrofulate
reductase

Fig. 829. Reduction of lol ate to letratiyflrofolate: A — folic acid inctudes pteridine ring attached
to para-ami nobenzoic acid iPABA}, and glutamate; B - tetra hydrofolate (H4-foEate); 0 — Dy liver
enzymes (fa late reductase and dinydrofoiate reductase, coenzyme — NADPH: fotate is successively
reduced to dihydrofolate and tetrahydrofoiate

biological yuf?ciio?i of tetmAydrofofaie. Most forms of tetrahydrofoiate arc

interconvertible and serve as donors of one-carbon units (one-carbon groups) in a
variety of metabolic reactions (Fig. 8.30).
444 Chapter 8. Nitrogen metabolism

Oxidation State Structure Name

Most reduced —CHa Methyl

Intermediate —CH?— Methylene

Most oxidized — CHO Formyt

— CHNH Formimino
—CH— Methenyl

Fig. 8.30. A key feature of H+-folate is that it can carry a variety of one-cartoon groups

The primary’ source of one-carbon units for letrahydrofolaie is the carbon

removed in the conversion of serine to glycine and glycine cleavage, producing N5,
Nltt-methylene-telrahydrofolate. Tetrahydrofolate is the substrate for the synthesis of
coenzymes, participating in one-carbon radical transfer. These coenzymes participate
in the synthesis of purine and pyrimidine nitrogenous bases, dTMP, and glycine and
serine metabolism, methionine regeneration (Fig. 8.31).

NAD* NADH + H+

Fig. 8.31. One-carbon units biological function

Fb/afe deficiency is relatively common, and can be caused by imbalanced diet (low
in vegetables, fruits and meal), malabsorption of folic acid, hepatitis, cirrhosis and
other liver lesions inducing decreased activity of folate reductase. Folate deficiency
leads to damaged nucleotide biosynthesis and thus cell proliferation. As the precursors
for blood cells divide particularly rapidly, disturbances of the blood picture can occur,
with increased amounts of abnormal precursors for megalocytes
8.6. Special Pathways of Some Amino Acids: Phenylalanine Metabolism, Tetrabydrofolate... 445

rjcrr of sulfur drugs. Microorganisms arc able to synthesize folate

from t heir own components. The growth of microorganisms can, therefore, be inhibited
by which competitively inhibit the incorporation of 4-aminobcnzoatc
(PABA) into folate (Fig. 8.32). Sulfa drugs work as competitive inhibitors of specific
enzymes participating in Iblic acid synthesis, as well Since folate is not synthesized in
the human body (humans got folate from their diet), sulfonamides have no effect on
human metabolism.

c Sulfa drug - Anti metabolite

Para-aminobanzoic acid PaBa normal reaction

antimatabolite

Folic
acid

1st enzyme 2nd enzyme

Sutfonamide base structure ‘roolecT inhibited

A — FABA and sulfonamide base structure: B —

Fig. 8.32. Sulfonamides inhibit folic acid synthesis:
sulfadi mean structure; C — if me sulfa drug nas been substituted for the PABA, then tn e final enzyme
is inhibited and no folic acid is produced

Sulfur-containing amino acids. Transmethylation reactions

Methionine is an essential amino acid and important for protein synthesis:
methionyl-tUNA is the initiator in protein synthesis. Met is the source of sulfur
in cysteine synthesis. Methyl group of methionine is the mobile one-carbon
fragment used for methylation (or transmethylation) hence for the synthesis of
adrenaline, melatonin, choline, creatine, nucleotide methylation, inactivation and
detoxification in the liver. Methionine undergoes trans- and deamination like other
amino acids.
Methyl-group in an immediate donor of methyl-fragment is an active form —
S-adenisyfniethtoninefSAMc; Fig. 8.33).
446 Chapter 8. Nitrogen metabolism

Methionine +

Fig. 8.33. Formation of S-aflenisylmethioni ne (SAMe)

The structure — S^-CH, in SA Me is unstable and methyl group may easily be

cleaved and transferred to other compounds in transmethylation reactions:

R R - CHg

S-adeno sy im etnioni ne
(SAMe)
< J
Methylfransferase
S-adenosyl nomocy stei ne
(SAH)

Transfer of the methyl group from S-adcnosyl meth io nine to an acceptor yields
S-adenosylhomocysteine, which is subsequently broken down to homocysteine and
adenosine. The detachment of methyl-group and its transfer to acceptor is catalyzed
by methyltransferase (Fig. 8.34).

Synthesis:
adrenaline,
choirne,
creatine,
camrline. etc.

Detoxification
Homocysteine ol toxic metabolites
and medians#
Adenosine compounds
Serine
PLP
Cysteine
Homoserme

Fig. 8.34. Methionine metabolism

8.6. Special Pathways of Some Amino Acids: Phenylalanine MetaDoiism, Tetrafiyttrofolate... 447

Reaction of methylation is very important and occurs at a very intensive rate

that leads to a large expenditure of methionine. Methionine is regenerated from
homocysteine by the action of homocysteine melhyltransferase, donor of methyl group
is N5-me thyl-H4-folate (coenzyme in the reaction is BIZderivative — methylcobalamm).
Then methionine is reconvened to S-adenosyl methionine to complete an activated-
methyl cycle.
Cysteine is semi-essential because of methionine required for its synthesis.
Cysteine is particularly important for protein folding because thiol groups of
cysteine form covalent disulfide bonds. HS-group is found in some enzyme active
sites. Cysteine is used for glutathione, taurine, HS-CoA synthesis and the source
of sulfates in the body. Cysteine is catabolizcd to pyruvate and can be converted to
glucose.
In the body cysteine is synthesized from two amino acids: methionine furnishes
the sulfur atom and serine furnishes the carbon skeleton. Homocysteine undergoes
a reaction with serine to yield free cysteine in two subsequent reactions catalyzed by
PLP-requiring enzymes (sec above Fig. 834).
Homocystinuria is an inherited disorder caused by defects of homocysteine
metabolism. Asa result, the amino acids methionine and homocysteine cannot break
down. This causes high levels of both amino acids to build up throughout the body,
which can be toxic and cause damage to the nervous system. The most common form
of homocystinuria is characterized by myopia, dislocation of the lens al the front of
the eye. an increased risk of abnormal blood clotting, and brittle bones that arc prone
to fracture (osteoporosis) or other skeletal abnormalities. Some affected individuals
also have developmental delaysand learning problems. Nutritional (folic acid. Bl2and
B6 deficiency) and environmental factors, as well as specific medications, may worsen
this abnormality and provoke symptoms.

Transmethylation reactions

Creatine synthesis
Muscle celts and nerve tissue use a phosphorylated form of creatine to store energy
(Fig. 835). Of the approximate 120 grams of creatine that exists in the human body,
95% is localized in the skeletal muscle, and only a few grams arc localized in the
brain. Creatine phosphate (macroergic compound) is particularly important since
it replenishes ATP <a cellular unit of energy) without relying on oxygen. Normal
metabolism cannot produce energy as quickly as a muscle cell can use it, so an extra
storage source is needed. The phosphate group can be quickly transferred to ADP
448 Chapter 8. Nitrogen metabolism

to regenerate the ATP necessary tor muscle cont raction. The role of creatine in the
brain is essential. Energy synthesis in the brain via the cicatine/phosphocreatine/CK
system is thought to be involved in signal transduction in the central and peripheral
nerves, and in maintenanceof membrane potential.
Creatine phosphate can be broken down into creatinine, which is then excreted in
the urine.

NHZ* NH2+
I
C NH2
IC—NH2
NH argmne amfinine NH N — CH-,
1
CHS —
V
J. 1 SA Me SAH 1
CH3 A CHa
ATP
o.
ADP

Creative
COO" COO coo- kisia&e

Glycine Guank3inoacela!e Creatine Creatine pnosphale Creatinine

Fig. 8.35. Creatine synthesis

Creatinine clearance tests are used Lo measure kidney function: it maybe not within
the normal ranges due to kidney disease or damage . Creatinine excretion may be raised
in excessive food intake, intensive physical activity and increasing muscle bulk, as well
as diabetes mcllitus, hypothyroidism, acromegaly (pituitary' gland pathology), and
febrile slate. Urine creatinine excretion is reduced in starvation, vegetarian nutrition,
muscle dystrophy, hyperthyroidism, and anemia as well.
Creatine synthesis requires three amino acids: arginine, glycine and methionine. The
p roccss beg ins i n kid ney wi th the form ation ofgua nid i noacctale. T he n guanid i noaceta to
is transported into the liver where methylated by SAMc and converted to creatine.
Creatine is transported to muscles and the brain from the liver. Creatine kinase (CK)
catalyzes the reaction of creatine phosphate synthesis.

Phosphatidykholi ne synthesis
Phosphatidylcholine (lecithin) can be synthesized (Fig. 8.36) by the methylation
pathway that converts phosphatidylctha nolam inc to phosphatidylcholine, principally
in the liver. The methyl donor is S-adenosylniethionine. Phosphatidylcholine is
transported lo tissues by LDL. Phosphatidylcholine plays a particular function in the
metabolism of HDLand cholesterol reverse transport (Ch. 7.10).
8.6. Special Pathways of Some Amino Acids: Phenylalanine MetaDoiism, Tetranytfrofolate... 449

[EthanolamiFej
Phosphatidyl- y Pnosphatidylserine \
etnanOlamcne-serine 1
transferase A
O Phosphatidyl-
[ Sec ine) \ ■ 1 serine
* CH2—0—C—R1 cfecarboxyksse
O J
1 2
CH—0— C—R2
O
a
CH2—0 — P — 0 — CH?—■C4a— NHg \
O" COa
Phosphatidylemanoiamina

3SAM

Methyltransfera st?

3SAH

□ — GHZ— CH2“+N(CH3)3

Phospnatidyicnoiine

Fig. 8.36. Phosphatidyl enol ine synthesis pathway.Phospnatidylserine is decarboxylated to form

phosphatitfylamine (cephalin} which men methylated lo form phosphatidylcholine (lecithin)

Carnitine synthesis
Carnitine is the transporter of the acyl group into the mitochondria. Skeletal
muscles have 97% ofall carnitine in the body, they are dependent on carnitine provided
from endogenous synthesis (occurs in the liver and kidney, but not in skeletal or heart
muscles) or diet (meat and fish products). Carnitine levels were correlated with the
intake of essentia I amino acids, methionine, and lysine (as substrates of its endogenous
synthesis). Carnitine synthesis occurs by methylation of y-aminobutyric acid with
SAMc as methyl group donor; hydroxylation reaction ofy-buti robetai ne requires Fc2'
and vitamin C as reducing agent:
450 Chapter 8. Nitrogen metabolism

( Lysine )

T
y-Aminobutirate
3SAMs

3SAH
3 Methyl transferase

y-Butirobetaine

Carnitine

8.7. DECARBOXYLATION OF SOME AMINO ACIDS, SYNTHESIS

OF NEUROTRANSMITTERS AND HORMONES

Decarboxylation of amino acids

Some amino acids can undergo dccarboxylalion — the cleavage of carboxylic
group with CO? release. PLP-rcciuiring (B„) reactions arc catalyzed by decarboxylases:

r- ch—coo’ —:-----
■Amino • f*—ch2—nh2 + cot
acid
' * decarboxylase
nh3 (PlP) Amine
Amino acid
Products of this reaction arc amines with expressed biological activity: many
important neurotransmitters arc primary or secondary amines, derived from amino
acids. In addition, sonic polyamines that form complexes with I) NA (c.g., spermine,
spermidine) are derived from the amino acid ornithine, a component of the urea cycle.
Catecholamines synthesis and function see above in chapter 8.6.
1. Glutamate decarboxylation gives rise to y-aminobutyrate (GABA) which is the
main inhibitory neurotransmitter:

CO2

Glutamate y-aminobutyrate Succinate TCA

Glutamate (GABA)
decarboxylase
(PLP)
8.7. Decarboxylation of Some Ammo Acids, Synthesis of Neurotransmitters and Hormones 451

► GABA content in brain is ten limes more than other neurotransmitters. GABA
increases permeability of postsy naptic membranes for K *■ that induce deceleration
of nerve impulse, it increases respiration of nerve tissue and improves blood
supply of brain, as well. GABA inactivation occurs either by transamination and
conversion to succinate (TCA metabolite), or oxidation by monoamine oxidase
(MAO).
» GABA underproduction is associated with epileptic seizures.
► GABA analogs arc widely used in the treatment of epilepsy, damages of
cerebral circulation, mental retardation, depression, and hypertension. Levels
of GABA can. also be increased by administering inhibitors of the GABA-
degrading enzyme — (SABA aminotransferase, which is used as anticonvulsants
(e.g.T convulex).
2. Another important neurotransmitter, serotonin, is derived from tryptophan in
a two-step pathway (note that first reaction catalyzed by //rp/op/™
require tetrahydrobiopterin as the cofactor and molecular oxygen):

CO

5-hydroxy- ------ - ---------- * SEROTONIN

tryptophane Decai&oxytase
(PLP)

H4BP H2BP MELATONIN

► Serotonin stimulates smooth muscle contraction, regulates blood pressure, body

temperature, breathing, possesses antidepressant effect (•■ pleasure* hormone),
sleep patterns and mood, it provides welfare and reduce appetite. Serotonin takes
part in allergic reaction as well and formed in mast cells in a small amount.
> In pineal gland (epiphysis), serotonin is convened to melatonin which regulates
diurnal and season metabolic rhythms.
3. Histidine undergoes decarboxylation to histamine in mast cell of connective
tissue: it complexes with proteins and is reserved in secretory granules:

CO2

► Histamine produces many varied effects within the body. Histamine:

* stimulates stomach, juice and saliva secretion (gastric hormone);
* is involved in inflammatory response; it is powerful vasodilator, increases
permeability of capillaries inducing redness, tissue edema, and it has a central
role as a mediator of itching;
* provides local immune responses;
* decreases blood pressure but increases intracranial pressure inducing
headache;
452 Chapter 8. Nitrogen metabolism

* con tracts smooth muscle in lungs, and induces asphyxia (shortness of breath);
• acts as neuralransmliter (Fig. 8.37).

system

Fig. 8.37. Histamine functions

4. Acetylcholine is a stimulatory neurotransmitter of autonomic nervous system

(Fig. 8.38). Choline is synthesized from serine decarboxylation and the following
methylation by SA Me. Choline acetyltransferase catalyzes acetylcholine
formation:

coa 3SAMe 3SAH

J . _________ V J . Chorine
Serine decartojcylase Ethanolamine
(PLP) meffiyhransfera&e

0
rt3C HaC 1
H3C—N4-— Ch?—ch?—-Oh H.3C—n *■—Ch?—Ch?—O-— C — CHS
H3C H^C
ChoSfW AcetylchoBme

+
Choline
O acetyltransferase CoASh

CoA—s—c —“ ch3
Ace?yl-CoA

► Acetylcholine is the ncu rot ransm it ter that transmits signals between motor
nervesand skeletal muscles.
► Myasthenia is a chronic autoimmune neuromuscular disease that causes weakness
in the skeletal muscles, which arc responsible for breathing and moving parts
of the body, including the arms and legs. In most individuals with myasthenia
gravis, this is caused by antibodies to the acetylcholine receptor.
8.7. Decarboxylation of Some Ammo Acids, Synthesis of Neurotransmitters and Hormones 453

Acetylcholine (ACh) is made

Mitochondrion
from choline and acetyl CoA

'AceLyl Coa)
Acetyicholffw
In tre synapse, ACh is rapidly
Proven down by me enzyme
acetylcholinesterase {ACME)

Choline is transported Cac*

(3) into the axon terminal and
used to matte more ACh

Cholinergic
f receptor

Fig. 8.38. Acetylcholine function

Amines are inactivated by two ways: oxidation and methylation

Monoamines arc inactivated into aldehydes by amtae oxidases {monoamine
oxidases. family of flavoproteins) with deamination and simultaneous
oxidation. These types of inactivation are characteristic for dopamine, norepinephrine,
serotonin, and GABA:

fadh2 +H2O
-nh3
R— CHwaNH
Monoamine
Amins oxidase Imine Atdfifiyde H Carbonic On
acid

MAO inhibitors play, thereforeT an important rote in the pharmacological

interventions in neurotransmitter metabolism.
Histamine, epinephrine and norepinephrine arc inactivated by methylal ion with
SAMe participation, as well:

SAMs

The quantitative characteristics of the metabolism of amino acids learnt in this section
Creatinine contents in urine: 8.8—17.6 mMol/L
Creatinine excretion: 1—2 g/24 h (directly proporlional to muscle bulk
454 Chapter 8. Nitrogen metabolism

Review tests
1. To CCP metabolites the following arc? converted:
[ Glucose ]
Pyruvate < 1

Ma'a'.e-
CAC
Fumarate a-K&togJutarate ^Glutamate

Succinate Succmyl-CcA

Amino acids:
A. Lysine.
B. Threonine.
C. Asparagine.
D. Glycine.
E. Histidine.
2. From glycolysis and CCP metabolites the following arc synthesized:
Glucose

Phospnoglycerate^ —-11 |
*

Sucotfiate SuccinykCOA
8.7. Decarboxylation of Some Ammo Acids, Synthesis of Neurotransmitters and Hormones 455

Amino acids:
A. Alanine.
B. Glutamate.
C. Cysteine.
D. Asparagine.
E. Serine.
.1. Coenzymes I l^-folatc derivatives are used for:

Processes:
A. Purine nucleotide synthesis (C2).
B. Methionine regeneration.
C. Purine nucleotide synthesis (Ca).
D. Pyrimidine nucleotide synthesis (TTP).
E. Methylation reactions.
4. SA Me synthesis:

ch3 CH3

s
11
ch2

NHj

COOH

Participants of the react ion:

A. Transferase.
B. Methionine.
C. NAD* —w NADH + H'.
D. ATP —* PP, + Pr
E. Dehydrogenase.
456 Chapter 8. Nitrogen metabolism

5. Catecholamine synthesis the following lake part in:

Nervous tissue Epinephros

Epinephros

Enzymes and coenzymes:

A. M cl hyhra nsferase {SA Me).
B. Hydroxylase (H4-biopterine Fe2‘).
C. Decarboxylase (pyridoxal phosphate).
D. Hydroxylase (vitamin C).
E. MethylLransferasc (methyl-H4-folate).

Situational problems
1. Glucose synthesis was investigated in isolated hepatocytes. Different amino acids
were added to the cell culture and the rate of glucose formation was checking
in. In control experiment, the rate of gluconeogenesis was 0.15 pmol/min of
glucose forma lion. Wien alanine, proline and glutamic acid were introduced to
cell culture, the rale of gluconeogenesis was increased to 0.17-0.18 pmol/min,
but the adding of lysine or leucine had no effect. Why the introduction of certain
amino acids increases the rate of *n.cw glucose* formation? For the answer:
a) write down the process taking alanine as the substrate:
b) specify the group to which used in experiment amino acids arc related to.
c) write dowrn the scheme of gluconeogenesis originated from these amino acids.
2. In volunteer group starving more than 2 days, blood glucose concentration
reduced till 60 mg/dl. Il was revealed that glucose in blood increases to 130 mg/dl
after glutamate and aspartate injection. What cause mentioned changes? For the
answer:
a) write down the first reaction in catabolism ofGlu and Asp;
b) specify the group due to the fate of nitrogen-free residue they belong to;
c) write down the scheme of these amino acid nitrogen-free residues usage in
reduced blood glucose levels.
3. A 2-year-old child was taken to the hospital. His mother said that he vomited
frequently, especially after feedings. The child’s weigh Land physical development
were below normal. His hair, although dark, contained patches of while. A urine
sample treated with ferric chloride (FcCI3) gave a green color characteristic of
the presence of pho nylpyru vale. Quantitative analysis of urine samples revealed
the presence of phenylalanine 7.0 pMol/L (0.01 in health), phe nylpyru vale
8.7. Decarboxylation of Some Ammo Acids, Synthesis of Neurotransmitters and Hormones 457

4.8 pMol/L (Din health) and phenyl lactate (Din health). Suggest, which enzyme
might be deficient i n this child? Why does the abnormal pathway come into play
under those conditions?
a) designate the disorder;
b) write down the reaction of damaged pathway; explain the appearance of
phenylalanine in the urine in the large amounts;
c) write down the scheme of an alternative metabolic pathway of the substrate;
d) explain the presence of white patches in the boy’s hair;
e) propose treatment.
4. The mother of a 4-month-old female baby attended in the well-baby clinic with
the complaint of black staining of the diaper after few minutes of urination.
The baby was born, of a non-consanguineous marriage, healthy and breast fed.
Mother noticed that stain first at the age of two and half months. The urine,
when kept in a test tube for two hours, turned black. What is the reason of
present symptom? What compound was revealed in increased concentration in
laboratory examination of urine? For the answer:
a) name compounds which, impart dark color to the urine of patient, point out
the disorder this symptom is observed in;
b) write down the scheme of metabolic pathway in which named! component is
intermediary metabolite, specify enzymesand cofactors;
c) specify the reaction in the scheme decreased rate of which causes the disorder;
d) presume. wrhat type of mutation can. lead to this pathology.
5. Amino acid methionine is used as medicine due to its lipotropic effect (^removes*
fat excess from the liver) in initial stages of liver cirrhosis, toxic liver lesions,
and chronic alcoholism. Methionine treatment reduces cholesterol contents
in blood and i ncreases the level of phospholipids. What arc mechanisms of the
therapeutic effect of methionine? For the answer
a) specify methionine functions in the human body:
b) write down the reaction of methionine activation;
c) name particles which arc formed to transport synthesized lipids from the liver
into the blood and then to tissues; describe their composition and metabolism;
d) point out the compound, which is necessary to build chosen lipid transport
forms. Specify the role of methionine in its synthesis. Present an appropriate
scheme;
c) specify, which particles have antiatherogenic activity and reduce the risk of
atherosclerosis development.
6. The doctor prescribed vitamin Bq (U(„ Iblic acid) in complex with Bpto infant
suffering megaloblastic anemia to stimulate erythropoiesis. What is the action
mechanism of these vitamins? is prescription expedience? Why severe liver
disorders can lead to B,,deficiency symptoms? For the answer:
a) write down the scheme of vitamin Byconversion to coenzyme, specify
enzymes, name amino acids which arc donors of one-carbon group:
b) enumerate derivatives of this coenzyme;
c) point out the processes these derivatives take place in;
d) answer the main question of the problem.
458 Chapter 8. Nitrogen metabolism

7. Sulfonamides arc used to treat bacterial infections. Why these medicines do

possess antibacterial effect, but do not manifest the cytostatic effect on. human
body cells? For the answer
a) present the scheme of folic acid structure. Name the main parts of folic acid
molecule-
b) write down the scheme of folic acid coenzymes formation;
c) present the general structure of sulfonamides and explain their action
mechanism;
d) specify processes which arc disturbed in bacterial cells in. sulfonamides
administration;
e) explain the inefficiency of sulfonamides in medium with high content of
p-aminobenzoic acid (PAUA) (pus, tissue degradation products).
8. Why are histamine and serotonin contents increased in the site of inflammatory?
Explain the importance of this fact recollecting the biological function of these
compounds. For the answer;
a Hist biological functions of histamine and serotonin in the human body:
b) draw the reactions of histamine and serotonin synthesis, point out enzymes,
localization, and necessity of coenzymes;
c) specify the pathways of biological amines inactivation and draw the schemes
of histamine and serotonin inactivation.
9. For stabilization of disturbed brain functions in mental disease nootropic agents
arc widely used in elderly persons and children. The main medicine of this group
is piracetam {nootropil) which is synthetic analogous of GABA, besides this it
is capable to accelerate dopamine synthesis and increases norepinephrine level.
What is the mechanism of these drugs activity in brain disorders? For the answer:
a) recollect biological functions of GABA, dopamine, norepinephrine:
b} draw the reaction of GABA, dopamine, and norepinephrine synthesis, point
out enzymes and coenzymes;
c) draw the reaction of GABA, dopamine, and norepinephrine inactivation and
specify enzymes.
10. Isoniazid (MAO inhibitor) is prescribed to the patient with Parkinson disease.
What docs cause this disorder? What is the mechanism of prescribed drag? For
the answer:
a) specify the neurotransmitter the disturbed formation ofwhich causes this disorder;
b) present the scheme of its synthesis and point out the most probable
enzymatic block;
c) write down the reaction catalyzed by MAC), give the full name of the enzyme,
specify coenzyme and explain the mechanism of isoniazid action.
11. Bottle-feeding newborns can suffer nervous system lesion linked with
Bf deficiency. What arc the biochemical mechanisms of given pathology
develop men L? For the answer:
a) explain the biological role ofBJn neurotransmitter and amino acid metabolism:
b) enumerate the main neurotransmitters and aniino acids which arc their
precursors;
c) give an example of reactions of known biological amines formation;
8.8. Special Products Derived from Amino Acids 459

d) specify pathways of neurotransmitter inactivation, write down reactions,

specify enzymes and coenzymes;
e) answer the main question of the problem.

8.8. SPECIAL PRODUCTS DERIVED FROM AMINO ACIDS

Synthesis and degradation of heme; jaundice

Heme synthesis and regulation
Heme is the prosthetic group of hemoglobin, myoglobin, cytochromes, catalase,
and peroxidase. Heme shuttles electrons between proteins as in mitochondrial
respiration or transports and stores O, as with the globins. It also contributes to the
red color found in muscles and blood. The prosthetic group consists of an iron atom in
the center of a protoporphyrin, which is composed of four pyrrole rings that arc linked
together by a methene bridge, four methyl groups, two vinyl groups and two propionic
acid side chains ( Fig. 8.39).

Fynrote - 5 member ring

Heme is a Pcnrphryin ring (telrapyrraie)

with a ctoetated iron Fe atom

Protoporphyrins contain 8 modified

side chains

3 types of modifications
I. 4 methyl groups, CH3
II. 2 vinyl groups, CH=CH3
ill. 2 propkmate groups, CH^-CHs-COOH

Fig. 8.39. Heme structure

The two starting materials in heme synthesis arc succinyl-CoA, derived from
the citric acid cycle in mitochondria and glycine. The product of the condensation
reaction between succinyl-Co A and glycine isa-aminolevulinate (ALA). This reaction
sequence is catalyzed by ALA synthase (pyridoxal phosphate-de pendent enzyme).
Synthesis of A LA occurs in. mitochondria (Fig. 8.40).
In the cytosol two molecules of ALA arc condensed by the enzyme ALA
dehydratase to form two molecules of water and one of porphobilinogen (PEG). ALA
dehydratase is a zinc-con lai ni ng enzyme and is sensitive to inhibition by lead, as can
occur in lead poisoning. The formation of a cyclic tctrapyrrolc — i.e., a porphyrin —
occurs by condensation of four molecules of PEG to form uroporphyrinogen HI by
the action of uroporphyrinogen HI synthase. Uroporphyrinogen has the pyrrole
rings connected by methylene bridges Uroporphyrinogen III is converted
460 Chapter 8. Nitrogen metabolism

Major sites of synthesis in the body:

SuCCinyi-GoA +■ Gycine CYTOPLASM
ALA BjrrtTtlHSe | - Bone marrow erythroid cells:
(ALA) about B5% of heme produced in
the body. Synthesis rate is
Aminolevulinic add — —■ Aminolevulinic add
relatively constant at all times

ALA dehydraiase
- Liver (-10%):
MITOCHONDRIA where cytochrome P450 is
synthesized. Synthesis is
Heme Rofpnobdmogen
up-regulated in response to
Hydf DwyiTl ei hylbilane
Fw.ocHslaiMS |fof) | syntfiase drug/alcohol metabolism

Protoporphyrin IX Hydroxymelhyi bilane Celltdar location:

P-fOioporptiyfjnuqert I UrDpofphyriiimj&fl III
ojc&ase | syrtdiase - Mitochondria:
Protoporphyrinogen IX Uroporphyrinogen m the initial reaction and the last
three steps
Coproporphyriirogefi f
oxidate 1

Coproporpriyrinogon ill
1 1 Uroporphyrinogen
decartozylase
— Copropaphyrinogen III
- Cytosol:
the intermediate 4 steps
Note: matire RBCs don’t have a
mitochondria and cannot make heme

Fig. 8.4(1 Heme synthesis pathway. Ta produce one molecule of heme, 8 molecules each cf glycine
and succinyl-CoA are required. A series of porphyrinogens are generated in sequence

io co pro porphyrinogen HI and then to protoporphyrin [X. Several steps arc involved in
this conversion. The final step in heme synthesis involves (he incorporation of ferrous
iron into protoporphyrin IX in a reaction catalyzed by ferrochelatase (heme synthase),
another mitochondrial enzyme.
85% of heme synthesis occurs in erythroid precursor cells in the bone marrow and
the majority of the remainder in hepatocytes. The porphyrinogens described above are
colorless, containing six extra hydrogen atoms as compared with the corresponding
colored porphyrins. However, the porphyrinogens arc readily auto-oxidized to their
respective colored porphyrins. These oxidations arc catalyzed by light and by the
porphyrins that are formed.
ALA synthase is the key regulatory enzyme in the biosynthesis of heme. Heme acts
as a negative regulator of the biosynthesis of ALA synthase: thus, the rate of synthesis
of ALA synthase increases greatly in the absence of heme and is diminished in its
presence. Fe*+ is an inductor of A LA synthesis in. reticulocytes.

The porphyrias are genetic disorders of heme metabolism

The porphyrias arc a group of disorders due to abnormalities in the pathway of
biosynthesis of heme; they can he or acquireJ. Depends on the organs or
cells that are most affected porphyrias may be predominantly either erythropoietic or
hepatic. Porphyrias can also be classified as acute or cutaneous on the basis of their
clinical features.
yfcw/e rntermMert porphyria is manifested by severe abdominal pain, pain in chest,
legs or back, constipation or diarrhea, nausea and vomiting, tingling, numbness,
8.8. Special Products Derived from Amino Acids 461

weakness or paralysis, red or brown urine, mental changes, such as anxiety, confusion,
hallucinations, disorientation or paranoia. CuAanrous /rorpAyrior can result in
permanent skin damage. The oxidation products, the corresponding porphyrin
derivatives, cause photosensitivity (Fig. 8.41), a reaction to visible light of about
400 nm. The porphyrins, when exposed to light of this wavelength, arc thought to
become ^excited- and then react with molecular oxygen to form oxygen radicals. These
latter species injure lysosomesand other organelles. Damaged lysosomes release their
degradative enzymes, causing variable degrees of skin damage. The skin, blisters can
become infected and when the skin heals alter cutaneous porphyria, it may have an
abnormal appearance and coloring, be fragile, or leave scars.

Fig. 8.41. Pnolosensitivity in porphyria

A large number of drugs (e.g., steroid hormones (estrogens), barbiturates,

diclofenac, sulfonamides, griscofulvin, etc.) Wire? /Acffl’yme Al.4 sy/j/Aose which is
the key regulatory enzyme of the heme biosynthetic pathway in the liver. High levels
of lead can affect heme metabolism by combining with HS-groups in enzymes such
as ferrochelatase and ALA dehydratase. This affects porphyrin metabolism and cause
porphyrias.
The photosensitivity (favoring nocturnal activities) and severe disfigurement
exhibited by some victims of congenital erythropoietic porphyria have led to the
suggestion that these individuals may have been the prototypes of so-called werewolves
(or vampires). No evidence to support this notion has been adduced.

Catabolism of hemeT bilirubin

Under physiologic conditions in the human adult, I -2* 10K erythrocytes arc
destroyed per hour. Thus, in I day, a 70-kg human turns over approximately 6 g of
hemoglobin. When hemoglobin is destroyed in the body, globin is degraded to its
constituent amino acids, which arc reused, and the iron of heme enters the iron pool,
also for reuse. The iron-free porphyrin portion of heme is degraded, as well, mainly
in the reticuloendothelial cells of the liver, spleen, and bone marrow. The catabolism
of heme from all of the heme proteins appears to be carried out in the microsomal
fractions of cells by a complex enzyme system called heme oxygenase (HO).
462 Chapter 8. Nitrogen metabolism

The heme oxygenase system issubstra/e-induc/ife. Oxygen is added to the a-methenyl

bridge between pyrroles of die porphyrin (thus, formation of verdoglobin t. then
ferric ion is released, carbon monoxide is produced, and biliverdin (green) results
from the splitting of the tetrapyrrole ring. An enzyme called biliverdin reductase
(BVR) produces bilirubin, a yellow pigment (Fig. 8.42). h is estimated /jW I g o/
hemoglobin yields 35 mg of bilirubin. The daily bilirubin formation in human adults
is approximately 250-350 mg, deriving mainly ITom hemoglobin and from various
other heme proteins such as cytochrome P The chemical conversion of home
to bilirubin by reticuloendothelial cells can be observed in. vivo as the purple color
of the heme in a hematoma is slowly convened to the yellow pigment of bilirubin.

Fig. 8.42. Heme catabolism in reticuloendolelia I system

8.8. Special Products Derived from Amino Acids 463

Pay attention that formed products have evident biological effects.

The further metabolism of bilirubin occurs primarily in the liver. Il can. be di vided
into three processes: (1) uptake of bilirubin by liver parenchymal cells, (2) conjugation
of bilirubin with glucuronatc in the endoplasmic reticulum, and (3) secretion of
conjugated bilirubin into the bile (Fig. 8.43).

E xtravascular w intfawascuJar hetnoly&rs

I
tMconjugaied bilirubin UrotJifinaqeft
Blood
Hepatic sinusoid
LMcortfugaDea bilirubin
iianiporlod

Hepatocyte icqugJKd to
gtKWDnic and

Cunjutated thliubin Uraniltnageri

UdDirKjgen
foddered in

bancral
Friases
Conju^aied bilirubin - Urobilinogen

Small intestine

Fig. 8.43. Heme degradation Heine is degraded to Li limb in carried in me blood try albumin, conjugated
to form tn e di glucuronide in tn e liver, and excreted in me bile, in tne intestine bilirubin is converted to
urobilinogen by fecal microflora

Bilirubin is only sparingly soluble in water, but its solubility in plasma is increased
by noncovalent binding to albumin. Each molecule of albumin appears to have
one high-affinity site and one low-affinity site for bilirubin. In 100 mL of plasma,
approximately 25 mg of bilirubin can be Lightly bound to albumin at its high affinity
site. Bilirubin in excess of this quantity can be bound only loosely and thus can easily
be detached and diffuse into tissues. A number of compounds such as antibiotics and
other drugs compete with bilirubin for the high-affinity binding site on albumin. Thus,
these compounds can displace bilirubin from albumin and have significant clinical
effects.
in the liver, the bilirubin is removed from albumin and taken up at the sinusoidal
surface of the hepatocytes by a carrier-mediated saturable system. Once bilirubin
enters the hepatocytes, it can bind to certain cytosolic proteins, which help to keep it
solubilized prior to conjugation. They may also help to prevent the efflux of bilirubin
back into the blood stream.
464 Chapter 8. Nitrogen metabolism

Bilirubin is hydrophobic and would persist in cells (c.g.. bound to lipids) if not
rendered water-soluble. Conjugation of bilirubin with glucuronic acid occurs in the
liver. Hepatocytes convert bilirubin to a hydrophilic form (water solublek which is
readily excreted in the bile, by adding glucuronic acid molecules to it. This process is
called conjugation. The conjugation of bilirubin is catalyzed by a specific glucuronyl
transferase. The enzyme is mainly located in the endoplasmic reticulum, uses UDP-
glucuronic acid as the glucuronyl donor, and is referred to as bilirubin-UGT. Bilirubin
monoglucuronide is an intermediate and is subsequently converted to the diglucuronide
referred to as conjugated bilirubin t Fig. 8.44}.

’Conjugation

BdifLibin
monoglucuronidg .

Bilrfufin
diglucuronidfl

Fig. 8.44. Formation of bilirubin monoglucuronide and bilirubin diglucuronide (direct) in hepatocytes.
UDPQA - UOP-glucuronic acid

Most of the bilirubi n excreted in the bile of mammals is in the form of bilirubin
diglucuronide. Bilirubin-UGT activity can be. induced by a number of clinically useful
drugs, including phenobarbital.
Secretion of conjugated bilirubin into the bite occurs by an active transport
mechanism, which is probably rate-limiting for the entire process of hepatic bilirubin
metabolism. The hepatic transport of conjugated bilirubin into the bile is inducible by
those same drugs that arc capable of inducing the conjugation of bilirubin.
As the conjugated bilirubin reaches the terminal ileum and the large intestine, the
glucuronides arc removed by specific bacterial enzymes (glucuronidases), and the
pigment is subsequently reduced by the fecal flora to a group of colorless lelrapyrrolic
compounds called urobilinogen. In the terminal ileum and large intestine, a small
fraction ofthe urobilinogen is reabsorbed and rccxcrctcd through die liver to constitute
the e/f/cro/jcpij/fc MrofcilfHogef? cycle. A small part of urobilin is excreted by kidney
(1,-2 mg/24 h). Under abnormal conditions, particularly when excessive bile pigment
is formed or liver disease interferes with this im rahepa tic cycle, increased urobilinogen
may also be excreted in the urine. Normally, most of the colorless urobilinogen formed
8.8. Special Products Derived from Amino Acids 465

in the colon by the fecal flora arc oxidized there to urobilin (or stercobilin - colored
com pounds) and arc excreted in the feces. The darkening of feces upon standing in
air is due to the oxidation of residual urobilinogen to urobilin.
A method for quantitatively assaying the bilirubin content of the scrum was first
devised by van den Bergh by application of Ehrlich's test for bilirubin. The Ehrlich
reaction is based on the coupling of Ehrlich’s diazo reagent and bilirubin to produce
a reddish-purple azo compound. The form of bilirubin that would react without the
addition of methanol was thus termed -direct-reacting * To that form of bilirubin
which could be measured only after the addition of methanol, the term "indirect-
reacting* was applied. The indirect bilirubin is - free* (unconjugated) bilirubin its route
to the liver from the reticuloendothelial tissues, where the bilirubin was originally
produced by the breakdown of heme porphyrins. Conjugated bilirubin, being water-
soluble, can react directly with the diazo reagent, so that she :■ direct bilirubin-.- is
actually a bilirubin conjugate (bilirubin glucuronide). Because of its hydrophobicity,
only unconjugated bilirubin can cross the blood-brain barrier into the eeniral nervous
system: thus, encephalopathy due to hyperbilirubinemia (kernicterus ) can occur only
in connection with unconjugated bilirubin. On the other hand, because of its water­
solubility, only conjugated bilirubin can appear in urine (choluria).

Hy perbili ru bittern ia causes jaundice

When bilirubin in the blood exceeds I mg/dL (17.1 pmoi/L), hyperbilirubinemia
exists. Hyperbilirubinemia may be due to the /jrac/wc/tow of more bilirubin than the
normal liver can excrete, or it may result from the failure of a damaged liver to exc/rto
bilirubin produced in normal amounts. In the absence of hepatic damage, obstruction
of the excretory7 of the liver — by preventing the excretion of bilirubin - will also
cause hyperbilirubinemia (Table 8.3).
Table 8.3 Diagnosis of jaundice types

Types oi jaundice Pre hepatic Hepatic Poa! hepatic

Type of bilirubin elevated Unconjugated bilirubin Bota conjugated & C-zoiugaled bilirubin
unconjugated bilirub- -

Serum bilirubin incfireci positive Biphasic Direct positive

Van den Bergh tesl

Urine
’ r Conj. Bi&ubin Absent ++ + * +■
,z UrobSncaen +++ + early, Obst -dec. Absent
Bite salt “ Abaani + ++

Urins color Normal ■ Acholuric Darc - Choluric Darc - Choluric

Stool color Dark brown colour N / decreased Qay-colored stools

AST a ALT Normal Very high inc.

ALP Levels Normal 2-3 limes increased 10-12 limes inc.

466 Chapter 8. Nitrogen metabolism

Hemolytic (prehepatic) jaundice is a consequence of accelerated erythrocyte

hemolysis that occurs in genetic defects of glucose -6- phospha to dehydrogenase,
pyruvate kinase or proteins of erythrocyte plasmatic membrane, strong oxidants
poisoning, and hemotransfusion of incompatible blood. Increased production of
bilirubin results in the increased blood level of indirect bilirubin (2-3 times than in the
norm). Urobilin and stercobilin contents in urine and feces arc elevated respectively.
Hepaticjaundice accompanies different types ofhepatitis, cirrhosis, etc. Dysfunction
of the hepatic cells results in decreased ability of hepatocytes to uptake bilirubin from
blood and defective conjugation and secretion, that leads to increased blood levels
of both indirect and direct bilirubin, but decreased contents of products of heme
catabolism in urine and feces. Direct bilirubin is excreted by kidney and imparts a dark
brown color to urine, but feces become light-colored due to decreased stercobilin.
Mechanical (obstructive, post-hepatic) jaundice is due to problems occurring
ow/siJe the liver that affect biliary (low within the gall bladder, bile duct, or biliary
papilla, in duodenum, i.c.. cholestasis and may be caused by obstruction of bile ducts
by stones, tumors of pancreas or postoperative adhesions. Blood level direct bilirubin
is elevated, direct bilirubin excreted by kidney dyeing urine to brown. Light-colored
stools arc observed due to stercobilin absence.
In all these situations, bilirubin accumulates in the blood, and when it reaches
a certain concentration (approximately 2-2.5 mg/dL) it diffuses into the tissues,
which then become yellow. That condition is called jaundice or icterus (Fig. 8.45).
In clinical studicsofjaundicc. the measurement of bilirubin in the scrum is of great
value.

Fia 8.45. Sclera icteritiousness

Neonatal «physiological jaundice*. This transient condition is the most common

cause of unconjugatcd hyperbilirubinemia, k results from accelerated hemolysis
around the lime of birth and an immature hepatic system for the uptake, conjugation,
and secretion of bil irubin. Not only is the bilirubin-UGT activity reduced. but there
probably is reduced synthesis of the substrate for that enzyme. LI DP-glucuronic acid.
8.8. Special Products Derived from Amino Acids 4&7

Since the increased amount of bilirubin is uuconjugated, it is capable of penetrating

the blood-brain barrier when its concentration in plasma exceeds that which can be
lightly bound by albumin (20-25 mg/dL). This can result in a hyperbilirubinemic
toxic encephalopathy, or kern icterus, which can cause mental retardation. Because of
die recognized inducibility of this bilirubin-metabolizing system, phenobarbital has
been administered to jaundiced neonates and is effective in this disorder. In addition,
exposure to blue light (phototherapy) promotes the hepatic excretion of unconjugated
bilirubin by convening some of the bilirubin to a. variety of water-soluble derivatives
that arc excreted in the bile.

Synthesis and degradation of purine and pyrimidine nucleotides.

Disorders of these metabolic pathways
Human tissues can. synthesize purines and pyrimidines from amphibolic
intermediates. Ingested nucleic acids and nucleotides, which, therefore, arc dietary
noncsscntial, arc degraded in the intestinal tract to mononucleotides, membrane­
bound nucleotidase enzymes in the epithelial cells of the ileum digest the nucleotides
io sugar, base, and phosphate, which arc absorbed.

Synthesis of purine and pyrimidine nucleotides

Purine and pyrimidine nucleotides arc synthesized in vivo at rates consistent
with physiologic needs. Intracellular mechanisms sense and regulate the pool sizes
of nucleotide triphosphates (NTPs), which rise during growth or tissue regeneration
when cells arc rapidly dividing. Two types of pathways lead to nucleoli des biosynthesis:
the A- worn synthesis and the De novo synthesis of nucleotides begins
with their metabolic precursors: amino acids, ribose 5-phosphate, COj, and NH3.
Salvage pathways recycle the free bases and nucleosides released from nucleic acid
breakdown. Both types of pathways arc important in cellular metabolism and both arc
presented in this section.

De novo synthesis

The formation of 5’-phosphoribosyl-1-pyrophosphate (PRPP)

An intermediate of major significance in nucleotide metabolism 1-phosphoribosy I-
5-pyrophosphate (PRPP) is formed from the ribose 5-phosphate and adenosine
triphosphate (ATP):

o o

Oh Oh Oh Oh O O

Ritxise- 5-ptiosptoaLe 5-phosphoribosyl- 1 pyrophosf)tia1.e {PRPP}

468 Chapter 8. Nitrogen metabolism

PR PP is involved in the de novo synthesis ofboth pyrimidine and purine nucleotides,

in the salvage pathways for both pyrimidine and purine bases, in. nucleotide Coenzyme
biosynthesis.

Biosynthesis of purine nucleotides

In the first step of the pathway, an amino group donated by glutamine is attached
at CL of PRPP (enzyme - amidotransferase). The resulting 5-phosphoribosyl-l-
amine is highly unstable with a half-life of 30 seconds at pH 7,5. The purine ring is
subsequently built up on this structure (Fig. 8.46):

Gin Glu

Amidotransferase

Pnospnoribosyi pyrophosphate ^PRPP) Phospnoribosy I-1-amine

The first intermediate with a complete purine ring is inosine-5"-monophosphate

(IMP). The enzymes of 1 MP synthesis appear to be organized as large muhienzymc
complex in the cell:

Fig. 8.46. The origin of purine ring atoms. Pay attention: nitrogen atom in NH?-group of
5-pnospnoriDosyl-t-amine is N,. in purine ring. Nr N^ — amide nitrogen of Gin; C4, Cr Nr — Glycine;
Cy, CE—metnenyl-H4-foiate(8};Jformyl-M(-foiate{2); Cs — COZ; N, — Aspartate

Synthesis of AMP and GMP

Conversion of IM P to AM P requires the insertion of an amino group derived from
.aspartate (Fig. 8.47): this takes place in two reactions. A crucial difference is that
GTP. rather than ATP. is the source of the high-energy phosphate in synthesizing
adenylosuccinate. GMP is formed by the NAD'-requiring oxidation of inosinate at
C-2, followed by addition of an amino group derived from glutamine; ATP is cleaved
io AMP and PPi in the final step (Fig. 8.47).
8.8. Special Products Derived from Amino Acids 469

H
“OOC- - CHy—C—- COO"

[XanUiylate (XMPJJ [Guanylate {GMP)J

Fig. 8.47. AMP and GMP synthesis: 1 — adenylosuccinate synthetase; 2 — adenylosuccinate lyase;
3 - IMP dehydrogenase; 4 — GMP synthase -iXMP glutamine amidotransferases)

Synthesis of nucleoside diphosphate and nucleoside triphosphate forms occurs by

nucleoside monophosphate kinase (NMP kinase) and nucleoside diphosphate kinase
NDP kinase):
► ATP -I- A M P - 2ADP; bv enzyme adenvM kinase (AM P kinase);
» GM P + ATP -* G DP + AD Pi by enzyme G M P kinase;
» NTPd + NDPa - NDPd + NTPa; by the ubiquitous enzyme.
d — donor: a — acceptor
Ubiquitous enzyme — nucleoside diphosphate kinase is not specific for the base
(purines or pyrimidines) or the sugar (ribose or deoxyribose); although the donor
(NTPd) is almost invariably ATP. because it is present in higher concentration than
other nucleoside triphosphates under aerobic conditions. Pay attention that ATP is
formed by glycolysis and oxidative phosphorylation in mitochondria.

Pyrimidine nucleotide synthesis

De wow pyrimidine nucleotide biosynthesis (Fig. 8.48) proceeds in a somewhat
different manner from purine nucleotide synthesis; the six-membered pyrimidine
ring is made first and then attached to ribosc-5-phosphalc. Required in this process is
carbamoyl phosphate, also an intermediate in the urea cycle. However, the carbamoyl
phosphate required in urea synthesis is made in mitochondria by carbamoyl phosphate
synthetase 1, whereas the carbamoyl phosphate required in pyrimidine biosynthesis
is made in the cytosol by a di fie rent form of the enzyme, carbamoyl phosphate
synthetase II.
470 Chapter 8. Nitrogen metabolism

CQ2+ Glutamine + ATP

CariHrtioyi

M3N 3
E pnotphace

O
SyffltldM II

ncHz

O—(?) &C-H Aspartate

XH3N ^Qtfinscaitjamoylaase

Carbamoyl Aspsrlk acid Carbamoyl aspartic acid (CAA| Dtfiydroorolic add IDHC'AI
prtosprtale (CAP)
WAD"-

DifiydroorotaEe
aEe
«e
deliytfrogenase ■.*•

NADH + H+-

CO2
V®

■j ■: I: z ,-lic acid
decarboxylase

LIMP OMP

NADPH * H” NADP*

dUDP
Ribonucleotide
reductase

jx“NS,Nl0-Me1hilene H4 Folate

© [ THymidylaEe syttdiase

P* H2 folate

TMP

Fig. 8.48. Conversion of carbamoyl phosphate ana aspartate to UMP. Not mentioned enzymes in me
figure: 7 — NMP kinase: 8 — NDP kinase (ubiquitous enzyme): 11 — phosphatase. Pay attention —
cseoxyribonucseatides are syntnesizecf in diphosphate form, OllMP is me substrate fordTMP formation
8.8. Special Products Derived from Amino Acids 471

Carbamoyl phosphate reads with aspartate to yield jV-carbamoy I aspartate in the

first committed step of pyrimidine biosynthesis. This reaction is catalyzed by aspartate
transcarbamoylase. By removal of water from jV-ca.rbamoylaspartatc, a reaction
catalyzed by dihydroorolase, the pyrimidine ring is closed to form L-dihydroorotate.
This compound is oxidized to the pyrimidine derivative o rotate, a reaction in which
NAD is the electron acceptor.
Once orotatc is formed, the ribose 5-phosphate side chain, provided once
again by PRPP. is attached to yield orotidylate (orotidine monophosphate. OMP).
Orotidylate is then decarboxylated to uridylate (uridine monophosphate, UMP), which
is phosphorylated to UTP (by N MP kinase and ubiquitous enzyme, sec above).
In human tissue, the six enzymes arc encoded by three genes. One gene codes for
a multifunctional polypeptide (CAD enzyme) that is located in the cytosol and has
carbamoyl phosphate synthetase II, aspartate transcarbamoylase, and dihydroorolase
activity. The native enzyme exists as multiples of three subunits. The second gene
codes for dihydroorotate dehydrogenase, which is located on the outer side of the
inner mitochondrial membrane. Dihydroorotate, the product of CAI), passes freely
through the outer mitochondrial membrane and converts to orotatc. Orotatc readily
diffuses to the cytosol for conversion to UMP. The third gene codes lor another
multifunctional polypeptide known as UMP synthase: UMP synthase involves
onotate phospho ribosyl I ransferase and orotidylate (o roti di ne-5f-monophosphate)
decarboxylase activity. The use of multifunctional polypeptides is very efficient since
the intermediates neither accumulate nor become consumed in side reactions. They
are rapidly channeled without dissociation from the polypeptide.
Cytidine nucleotides arc formed front uridine nucleotides at the triphosphate level.
CTP is synthesized from UTP by transfer of the amide nitrogen of glutamine to C-4 of
the pyrimidine ring of UTP. This reaction requires ATP as an energy source.
Hereditary orotic aciduria caused by UMP synthase deficiency is characterized by
excessive excretion of orotic acid in urine because oft he inability to convert orotic acid
io UMP. Orotic acid accumulates, causing clinical manifestations of megaloblastic
anemia, orotic crystal I li ria and nephropathy, cardiac malformations, strabismus,
and recurrent infections. Mentioned symptoms occur due to the inability to provide
the normal rate of nucleic acid synthesis. These features respond to appropriate
pyrimidine replace me nt therapy, and most cases appear to have a good prognosis.

Synthesis of thymidine nucleotides (see above Fig. 8.4B)

Pc novo synthesis of thymidylic acid (TMP) occurs exclusively by methylation
of the C-5 of dUMP by thymidylate synthase. The methylene group of
mcthylenc methylone-H.J7 is the source of the methyl group, and H4-folate is
oxidized to H.-folate. For sustained synthesis, teirahydrofolatc must be regenerated
by dihydroFolate reductase

Synthesis of deoxyribonuc lent ides

Reduction of the 2'-hydroxyl of purine and pyrimidine ribonucleotides,
catalyzed by the ribonucleotide reductase complex, forms deoxy ribo nucleus ide
diphosphates (dNDPs) (Fig. 8.49). The enzyme complex is active only when cells
472 Chapter 8. Nitrogen metabolism

arc actively synthesizing DNA. Reduction of ribonucleoside diphosphates (NDPs)

to dcoxyribonuclcosi.de diphosphates (dNDPs) is subject to complex regulatory
controls that achieve balanced production of dcoxv ribonucleotides for the synthesis
of DNA.

Fig. 8.49. Formation of fleoxYribonucteolides. Reduction requires tniorefloxin (a small protein with
molecular weight 12 kD}, which is oxidized during me reduction of the ribose 2r-hydroxyl group of
the nucleotide dipn ospnate. rib ortuc leotide reductase, and NAD Ph. The immediate reductant, reduced
thio redoxim is produced by NADPn and thio redoxin reductase

Salvage synthesis
Free purine and pyrimidine bases arc constantly released in the cel! during the
metabolic degradation of nucleotides. Free purines arc, in large part, salvaged and
reused to make nucleotides, in a pathway much simpler than the de novo synthesis
described earlier. One of the primary salvage pathways consists of a single reaction
catalyzed by adenine phosphoribosyl transferase, in which free adenine reacts with
PRPP to yield the corresponding adenine nucleotide:

Adenine + PRPP—AMP + PPi.

Free guanine and hypoxanthine {the deamination product of adenine) arc salvaged
in the same way by hypoxanthine-guanine phosphoribosyhransferase:

Guanine + PRPP------- * GMP + PPi;

Hypoxanthine + PRPP------- ► 1MP + PPi.

A similar salvage pathway exists for pyrimidine bases in microorganisms, and

possibly in mammals.
A genetic lack of hypoxanthine-guanine phosphoribosyltransferase activity, seen
almost exclusively in male children, results in a bizarre set of symptoms called Lesch-
Nyhan syndrome. Children with this genetic disorder, which becomes manifest by the
age of 2 years, arc sometimes poorly coordinated and mentally retarded. In addition.
8.8. Special Products Derived from Amino Acids 473

they arc extremely hostile and show compulsive. self-destructive tendencies: they
mutilate themselves by biting off their fingers, toes, and lips.
The devastating effects of Leach-Nyhan syndrome illustrate the importance of the
salvage pathways. Hypoxanthine and guanine arise constantly from the breakdown
of nucleic acids. In the absence of hypoxanthinc-guaninc phospho ribosy II ransferase,
PR PP levels rise, and purines arc overproduced by the de novo pathway, resulting m
Ajgft levels of uric acid production andgouf-b'ke damage fa tissue. The brain is especially
dependent on the salvage pathways, and this may account for the central nervous
system damage in children with Lesch-Nyhan syndrome.

Purine and pyrimidine nucleotide biosynthesis is regulated by feedback inhibition

I. RegiJaf/on ofpyrimidine nuc/cofide synthesis (Pig. 8.5 0^
a) carbamoyl phosphate synthase fl is allostcrically inhibited by uridine
triphosphate -the end product of the pathway and by/wrf/ie wwc/co/Mfes, but
activated by PftPP;
b) as/iarra/e carbamoyl transferase is inhibited by CTP, but activated by ATP.
This regulation prevents the excessive synthesis of pyrimidine nucleotides, and
guaranteeing the balanced formation of all pyrimidine and purine nucleotides
required for nucleic acids synthesis.
Aspartate
+
Carbamoyl phosphale

ATCase T-i ”

ATP..... I
Carbamoyl aspartate
De novo pathway |
(steps 3-6) t
UMP

UDP
I

CTP synthetase
G7P...............

Fig. 8.50. Regulation ot pyrimidine nucleotide synthesis

11. Regidation ojpurine nuefeotide synthesis (Fig. 8.51.).

I. The committed step that is the formation of 5'phosphoribosyl I-amine by
allosteric enzyme amfdo/rons/erase. I.WP. GAfP, JjWParc negative modulators
of this enzyme:
474 Chapter 8. Nitrogen metabolism

2. Two mechanisms regulate the conversion of f M P to G M P and A M P: a) AMP

and G M P feedback-in hi bit adeny/osuccmate synthase and /AfP dehydrogenase
respectively; b) conversion of IM P to adenylosuccinate route to AM P requires
GTP, and conversion of xanthinylale (XMP) to GMP requires ATP. This
cross-regulation between the pathways of IMP metabolism thus serves to
decrease the synthesis of one purine nucleotide when there is a deficiency of
the other nucleotide. A reciprocal arrangement tends to balance the synthesis
of both ribonucleotides.
AM P and GMP also inhibit
wrhich. converts hypoxanthine and guanine to IMP and GMP, and GMP
fccdback-in hibits PJiJVglutamine amidotransjerase.

Ribose 5-pnospnate
PRPP synthetase
e--
I
5-pnospnor ibosyl 1 -pyjophospnais
(PRPP)
Glutamine phosphorytKssjl

e-1.....
amid'Otransferase

5-pnospncxibosyi 1-amine

t Adenylosuccinate

XMP * * Adenylosuccinate
1 ■ 1
! \ *
r GMP AMP **
jr
1 1
I I
GDP ADP
1 I
I
L GTP ATP

Fig. 8.51. The regulation of purine synthesis. PRPP synthetase has two distinct allosteric sites, one for
ADP, the other for GDP. Glutamine pnosphorioosyi aminotransferase contains adenine nucleotide and
guanine nucleotide-binding sites; me monophosphates are the most important, although the di- and
tri-pbosphates will also bind to and inhibit the enzyme. Adenylosuccinate synmetase is inhibited by
AMP: IMP dehydrogenase is inhibited by GMP

IIL 7'Ae PRPP synthase reaction. The rate of PRPP synthesis depends on the
availability of ribose 5-phosphate and on the activity of PRPP' synthase, an
enzyme sensitive to feedback inhibition by AMP, ADP, GMP, and GDP.

Purines degradation
Purine nucleotides arc degraded by a pathway in which they lose their phosphate
through the action of5'-nucleotidase. Adenylate yields adenosine, which is deaminated
to inosine by adenosine deaminase. and inosine is hydrolyzed to hypoxanthine (its
purine base) and ribose. Hypoxanthine is oxidized successively to xanthine and then
8.8. Special Products Derived from Amino Acids 475

urk acid by xanthine oxidase, a flavoprolein-cnzymc with an atom of molybdenum

and lour iron-sulfur centers in its prosthetic group. Molecular oxygen is the electron
acceptor in this complex reaction (Fig. 8.52).
GMP catabolism yields uric acid as an end product, as well. GMP is first
hydrolyzed to guanosine, which is then cleaved to free guanine. Guanine undergoes
hydrolytic removal of its amino group to yield xanthine, which is converted to uric acid
by xanthine oxidase.
NHi*

_____________ AMP
Atiteflylaie deaminarse
Nucleotidases
NH<+

Inosine _____________ Actenosine

Nucleoside Adenosine deaminase
phosphorylases

Xanthine Urate

Fig. 8.52. Purine nitrogenous bases degradation

The end product of purine catabolism in humans is uric acid. Both undissocialcd
uric acid and the monosodium salt (primary form in the blood) arc only sparingly
soluble and present in biologic fluids at the limit of its solubility: concentration of
urates in blood plasma is 0.1-0.3 mM/L; excretion with urine is 0.3-1.29 g/24h. A
very high concentration of urate in the blood (Ajpmrwemw) leads to a fairly com mon
group of diseases referred to as gout. Crystallization of urates in soft tissues and joins
forms deposits called tophi (tophus), which causes acute and chronic gouty arthritis
(Fig. 8.53). The joints become inflamed, painful and arthritic owing to the abnormal
deposition of sodium urate crystals. Gout occurs predominantly in males.
Gout is effectively treated by a combination of nutritional and drug therapies. Food
especially rich in nucleotides and nucleic acids such as liver or glandular products,
meal and fish, is withheld from the diet. Major alleviation of the symptoms is provided
by the drug alfopurinoL which inhibits xanthine oxidase (ewwpr/ftfve m/tibi/ion). Wien
xanthine oxidase is inhibited the excreted products of purine metabolism are xanthine
476 Chapter 8. Nitrogen metabolism

and hypoxanthine, which are more water-soluble than uric acid and less likely to form
crystalline deposits.

The limited solubility is not ordinarily a problem in urine unless the urine is
very acid or has high |Ca3'|. Urate salts coprccipitatc with calcium salts and can
form stones in kidneys: nephrolithiasis, which maybe reduced by alkalinization of
urine.
Xanthine oxidase deficiency due either to a genetic defect or to severe liver damage
in severe cases may exhibit xanthinuria and xanthine lithiasis.
.Adenosine deaminase deficiency leads to an accumulation of toxic purine
degradation by products, most potently affecting lymphocytes. Whilst most notable
effects arc on lymphocytes, other manifestations include skeletal abnormalities,
ncurodcvclopmcntal effects and pulmonary manifestations. Affected patients
present in early infancy, usually with persistent infection, or with pulmonary
insufficiency.
Purine nucleoside phosphorylase deficiency is one of several disorders that damage
the immune system and cause severe combined immunodeficiency (SCID). People with
SCID lack virtually all immune protection from foreign invaders such as bacteria,
viruses, and fungi. Affected individuals arc prone to repeated and persistent infections
that can be very serious or life-threatening. These infections arc often caused by
^opportunistic* organisms that ordinarily do not cause illness in people with a normal
immune system. Infants with SCID typically grow much more slowly than healthy
children and experience pneumonia, chronic diarrhea, and widespread skin rashes.
Without successful treatment to restore immune function, children with SCI D usually
do not survive past early childhood.
8.8. Special Products Derived from Amino Acids 477

Catabolism of pyrimidines
Human cells degrade pyrimidine nucleotides to their component bases. These
reactions, like those of purine nucleotides, occur through dcphosphorytaiion,
deamination, and gtycosidic bond cleavages. CMP, UMP and dTMP arc convened
into cytosine, uracil and thymine, ribose-1.-phosphate and deoxyribose-1-phosphate.
The Pyrimidine catabolism pathway generally lead to NH' production and: thus to urea
synthesis (Fig. 8.54).

H2N—-CH^— CHg— COOH HjN— d-t2— CH—COOH

'•Ha X.

p- alanine p-aminoisobutyrate '

Carnosine Excreted
or
Anserine

Fig. 8.54. Pyrimidiiie nitrogenous liases degradation. Cytosine is deaminated into uracil: reaction
is catalyzed Dy cytidine deaminase, uracil and thymine are converted into dinydrouracii and
dihydromymine Dy dinydrouracii dehydrogenase: one NADPH -t- H* is oxidized into NADP*.
Dinydrouracii and dihydromymine are converted into p-atanine and fkamino isobutyrate i cyclized
molecule is converted into linear Dy cleaving the covalent Doud at particular place}

The p-alaninc and p-amino isobutyrate is converted into malonic scmialdchyde

and methyl malonic scmialdchyde by transamination and then to succinyl-CoA.
p-alaninc is involved in muscular peptides anserine and carnosine synthesis. Bacteria
employ p-alanine into pantothenic acid synthesis thus 14SCoA.
Excretion of p-aminoisobutyralc increases in leukemia and severe x-ray radiation
exposure due to increased destruction of DN A. However, many persons of Chinese or
Japanese ancestry routinely excrete P-amino isobutyrate.
478 Chapter 8. Nitrogen metabolism

Nucleosides and nucleotides are widely used in clinical medicine and science
These derivatives:
* inhibit a particular enzyme of nucleotide or nucleic acid synthesis;
* arc synthetic analogous and inserted into DNA or KN A synthesis damaging the
complementation between, nitrogenous bases and polymerization of nucleic acid
chains.
The fluoropyrimidinc - 5-fluorouracil (5-FU) is an anti metabolite drug that is
widely used for the treatment of cancer, particularly for colorectal cancer. 5-FU exerts
its an ticancere fleets through inhibition of thymidylate synthase (TS)and incorporation
of its metabolites into RNA and DNA (Fig. 8.55: Fig. 8.56).

Synthesis

Fig. 8.55.5-fluorouracil (5-FU) is a thymidylate synthase (TS) inhibitor

Methotrexate is a structural analogous of folic acid and inhibits dihydro folate

reductase damaging purine nucleotide synthesis and conversation ofdUM P to dTMP
(Fig. 8.56).
Fluorouracil (5-FU)

dUMP---------------------------- * dTMP
Thymidylaie synlfiase

Fig. 8.5 B. Memotrexate and fluoro uracil media nism of action

8.8. Special Products Derived from Amino Acids 479

Acyclovir is widely used in the treatment ofherpes virus infections, particularly herpes
simplex virus (H SV) and varied la-zoster virus (VZV). Acyclovir (9-[2-hydroxymethyl |
guanine) triphosphate) competitively inhibits viral DNA polymerase by acting as
an analog to deoxyguanos inc triphosphate (dGTP). Incorporation of acyclovir
triphosphate into DNA results in chain termination since the absence ofa 3’ hydroxyl
group prevents the attachment of additional nucleosides.
Azido th vmidine (AZT) is an antiretroviral medication used to prevent and treat
H1V/AIDS. AZT is a thymidine analog. AZT works by selectively inhibiting HIV’s
reverse transcriptase (Fig. 8.57), the enzyme that the virus uses to make a DNA copy
of its RNA. Reverse transcription is necessary for the production, of HIV's double­
stranded DNA, which would be subsequently integrated into the genetic material of
the infected cell (where it is called a provirus). Cellular enzymes convert AZT into the
effective 5’-triphosphate form. The termination of HIV's forming DNA chains is the
specific factor in the inhibitory effect.

AZT (Zidovudine) Dkaeoxycyrkfine (ddC)

3'- azido-? J'-dideoxytfiymidine

Fig. 8.57. Mechanism of action of aziaotftymidiiie

The quantitative characteristics of the nwtaboGsm of amino acids learnt in this section
Blood uric acid concentration: 0.15-0.46 mMd/L
Uric acid excretion: 40D-600 mg/dl. (0.3—1.29 g/24h)

Review tests
I. The origin of C and N atoms in purine base:
480 Chapter 8. Nitrogen metabolism

The sourse of atom:

A. Ns — methyl - H+-folate.
B. M1* — formyl - H^-folalc.
C. N \ N|,J— methenyl — H4-folate.
D. Carboxylic group of Asp.
E. COr
2. Reduction of ribonucleoside diphosphates into deoxy-derivatives:

Component of the reaction:

A. H.-folate.
B. H,-folate.
C. Thio red oxi n-(SH)r
D. Th io redox in S—S.
E. Ribonucleotide reductase.
_T Synthesis of dTMP:

dUMP

dTMP

Enzyme:
A. Thymidylate synthase.
B. Thymidine kinase.
C. Serine hydroxymethyl transferase.
D. Di hydro folate reductase.
E. Ribonucleotide reductase.

Situational problems
I. The girl visited a. doctor with complaints of erythema, edema and itching which
was appeared on open parts of skin after the rest on the beach. Doctor found
out that the patient had taken the drug including sulfonamide not tong ago.
5-aminolevulinatc and porphobilinogen were found in blood, the urine was
8.8. Special Products Derived from Amino Acids 481

dyed to red. What could cause photo dermatosis in patient and what disorder
this girl sutlers from'? For the answer:
a) write down the first reactions and the following scheme of metabolic pathway
which intermediate products were found in the blood:
b) whaL enzyme synthesis was induced by sulfonamides, point out the mechanism
of its regulation;
c) explain the molecular mechanisms of occurred symptoms.
2. Two newborns with jaundice were prescribed to phototherapy. One newborn
showed the improvment in his state and symptoms of jaundice had been
disappeared. But phototherapy was useless lor the second baby and he was
prescribed phenobarbital. Unfortunately, this therapy was also inefficient,
moreover, encephalopathy symptoms became revealed. How to explain doctor
recommendations? What can cause jaundice in the second newborn? To answer
the question:
a) explain the cause of ^physio logical* jaundice in newborns:
b) specify the changes in contents of bilirubin in blood, stercobilin and urobilin
respectively in feces and urine in newborns:
c) explain the mechanism of phototherapy and phenobarbital: curative effect.
Write down the reaction which rate is increased by phenobarbital;
d) explain the results of the treatment.
3. A patient with high temperature 38.5 and clinically apparent icteric skin
and mucous tunics was delivered to the admitting office of hospital. Blood
concentration of both direct and indirect bilirubin was increased. Direct
bilirubin is found in urine, but urine urobilin and feces stercobilin were reduced.
What type ofjaundice patient suffered from? To answer the question:
a) present the scheme of indirect bilirubin formation;
b} write down the reaction of bilirubin conjugation:
c) point out the properties of direct and indirect bilirubin, explain the reasons
for indirect (Linconjugaicd) bilirubin toxicity;
d) specify enzymes, the activity ofwhich is of particular importance for diagnosis
for the liver damage. Describe the main principles of enzymod iagnostics.
4. A 67-ycar-old male attended the clinic with a painful 1-st metatarsophalangeal
joint, which had progressively worsened in pain, mobility and deformity. In
examination it was revealed redness, edema and increased local temperature.
Patient diet involves much amount of meat. Biochemical blood analysis revealed
uric acid concentration 0.51 mMol/L (normal for male 0.17-0.42 mMol/L).
What is the expected diagnosis in this case? For the answer:
a) write down the scheme of uric acid formation;
b) specify the mechanisms of presented symptoms development;
c) represent your diet recommendations to the patient;
d) propose the remedy prescribed by the doctor in case of this disorder.
5. Goul can be caused by supcractivalion of PRPP synthase, or partial deficiency
of hypoxanthine-guanine phospho ribosyl transferase. Why the change in these
enzyme activities can induce the development of this disorder? For the answer:
a) write down the scheme of the reactions catalyzed by these enzymes;
482 Chapter 8. Nitrogen metabolism

b) specify metabolic pathways these reactions take pari in;

c) answer the main question of the problem.
6. After intravenous administration of aspartate with N15 radioactive marker to
experimental animals, the labeled nitrogen is revealed in nucleic acids ofdifferent
tissues and organs. What atoms in purine and pyrimidine bases wilt include the
marker nitrogen? To answer the question, present the schemes of nucleotides
synthesis. Indicate the label Nlsin these schemes.
7. The methotrexate was prescribed to an oncological patient. The cytostatic effect
of this medicine is due to its influence on S-stage of the cell cycle. Why the block
of a particular reaction leads to the termination of tumor growth? To explanation
the molecular mechanism of antitumor action of methotrexate:
a) point out the biological role of folic acid;
b} enumerate folate derivatives and their function in the body:
cjdraw the scheme of folate synthesis; specify the reaction blocked by
methotrexate:
d) answer the main question olThe problem.
8. The Lesch-Nyhan syndrome in children is accompanied by a severe form of
hyperuricemia with tophi and urate calculus formation, and serious neurological
deviations. What enzyme activity lack is th is disorder linked with? For the answer:
a) write down the reaction which docs not occur in patients with noted disorder,
specify the enzyme the activity of which is lost in. these patients:
b} explain, what cause hyperuricemia and present the scheme of process
activation of which leads to accumulation of urates:
c) specify the medicine which can reduce the content of uric acid in the blood of
these children and explain its mechanism.
9. The diazo compound ()-(2-diazoacciyl)-L-scrine, known also as azascrine,
is a powerful inhibitor of glutamine amidotransferases. If growing cells arc
treated with azaserine, what intermediates of nucleotide biosynthesis would
accumulate? Explain and present an appropriate scheme.
 Chapter 9
MOLECULAR ENDOCRINOLOGY

9.1. Role of Hormones in Regulation of Metabolism and Functions

92. Structure, Synthesis and Basic Action of Hormones of the Hypothalamus a nd Pituitary
Gland
9.1 Structure, Synthesis and Basic Action of Principal Hormones Regulating Fuel
Metabolism
9.4., Hormonal Regulation of Fuel Metabolism at Normal Nutrition Rhythm
9.5. Metabolic Changes During Starvation
9.6. Metabolic Changes During Muscle Activity
9.7. Metabolic Changes in Hypo and Hypersecretion of Hormones
9JL Metabolic Changes in Diabetes Mellitus
9JL Actions of Hormones Regulating Sodium and Water Balance
9.10. Actions of Hormones that Affect Calcium and Phosphate Levels
9.11. Disorders of Calcium Metabolism

9.1. ROLE OF HORMONES IN REGULATION OF METABOLISM

AND FUNCTIONS
The classic definition of hormones of endocrine glands is that they arc organic
molecules that arc synthesized in specialized glands and secreted directly in the
bloodstream to acton target tissues. They function as chemical mediators to relay signals
about changes in the external and internal environment to various organs and tissues.
The cell response to the hormone action is very diverse and is determined by both the
chemical structure of the hormone and the type of cell that has the hormone action is
directed to. Hormones circulate in the plasma in very low concentrations {picomolar
to micromolar range). Therefore, to exert physiological c fleets of hormones on target
tissues, the cells must contain receptors on their plasma membranes or within their
interior, which recognize and bind the circulating hormones with high affinity and
specificity. The *life» of a hormone and duration of its physiological effect is dependant
on various factors, for example, the hormone concentration (that is determined by
the synthesis and inactivation rate, and the rate of excretion of hormones and their
metabolites from the body), its affinity for transport proteins tsteroid and thyroid
hormones arc carried along bloodstream combined with proteins), the number and
type of receptors on the target cells surface.
Hormones are synthesized in response to biochemical signals generated by various
systems of an organism. For example, insulin synthesis is regulated by the glucose
484 Chapter 9. Molecular endocrinology

concentration in blood, and PTH synthesis is regulated by the ionized calcium

concentration. For gonadal, adrenal, and thyroid hormones, control of their synthesis
is achieved by the hormonostatic function of the hypothalamic-pituitary axis. The
synthesis and secretion of steroid and thyroid hormones arc stimulated by external
and internal signals entering the central nervous system. These signals through
the neurons enter the hypothalamus to stimulate the synthesis of peptide releasing
hormones, libcrins and statins, which, respectively, stimulate or inhibit the synthesis
and secretion ofhormon.es of the anterior pituitary (Fig. 9.1).

External internal
©
Environmental I Centra nervous system
signals

-X^ .___
Pituitary
Tropic hormones

i
Endocrine Hands

Fig. 9.1. Hypotnalamic-pituitary-peripheral systems. 1 — synthesis and secretion of hormones is

stimulated Dy external and internal signals; 2 — signals along neurons enter trie hypothalamus, where
they stimulate the syntnesis and secretion of releasing hormones; 3 — releasing norm ones stimulate
(liberins) or inhibit (statins) the syntnesis and secretion of tropic hormones of me pituitary gland: 4 —
tropic hormones stimulate the synthesis and secretion of hormones of peripheral endocrine glands;
5 — hormones of the endocrine glands enter the bloodstream and interact with target cells; 6 —
change in the concentration of metabolites in target cells by the negative feedback mechanism inhibits
the syntnesis of hormones of the endocrine glands and the hypothalamus; 7 — the synthesis and
secretion of tropic hormones is suppressed by hormones of the endocrine glands; ■+ — stimulation
of the syntnesis and secretion of hormones: - — suppression of the synthesis and secretion of
hormones (negative feedback)
9.1. Role of Hormones in Regulation of Metabolism and Functions 485

Hormones of the anterior pituitary, called tropic hormones, stimulate the

synthesis and secretion of hormones of peripheral endocrine glands, which enter the
general circulation and interact with target ceils. Maintaining the level of hormones
in the body provides a negative feedback mechanism. Changing the concentration
of metabolites in target cells by the mechanism of negative feedback suppresses the
synthesis of hormones, acting on cither the endocrine glands or the hypothalamus.
Synthesis and secretion of tropic hormones arc suppressed by hormones of peripheral
glands. Such feedback loops operate in the regulation systems of the hormones of the
adrenal glands, the thyroid gland, and the sex glands. Not all endocrine glands arc
regulated in this way. Hormones of the posterior lobe of the pituitary gland (ADH
vasopressin and oxytocin) arc synthesized in the hypothalamus as precursors and
stored in the granules of the terminal axons of the neurohypophysis. The secretion
of pancreatic hormones (insulin and glucagon) is directly dependent on the blood
glucose concentration.
Major human hormones arc classified according to their chemical structure,
biological functions and mechanism of the action. Hormones may be divided into
3 groups by chemical composition:
► peptide (or protein):
► steroid:
► non-peptide amino acid derivatives (Table 9.1).
Table 9.1 Classification d hormones according to chemical structure

Adrenocorticotropic hormone (ACTH, adrenacarticotropin or Aldosterone Epinephrine

corticotropin) Cortisol Norepinephrine
Growth hormone <Somatotropin) Calcitriol Triiodothyronine
Growth hormone-releasing hormone (GHRH) Testosterone
Somatoctatin (SS) Estradiol Thyroxine
Ttiyrotropin-releasing hormone (TRH) Progesterone Melatonin
Thyroid-stimulating hormone (TSH).or
Thyrotropin or thyrotropic hormone)
Prolactin (PRL, luteotroprc hormone or luteotropin)
Lute inking hormone (LH)
Follicle-stimulating hormone (ESH)
Gonadotropin-releasing hormone 4GnRH)
Melanocyte stimulating hormone
Human chorionic gonadotropin (hCG)
Antidiuretrc hormone (ADH, vasopressin)
Oxytocin
Parathyroid hormone (PTH)
Calcitonin
Thyrotropin-releasing hormone (TRH)
Insulin
Glucagon

According to biological functions, hormones can be divided into several groups

(Table 9-2). The same hormones can perform di fie rent lime lions. For example,
epinephrine regulates the metabolism of fats and carbohydrates and, in addition,
regulates the heart rale, blood pressure and smooth muscle contraction. Cortisol
stimulates gluconeogenesis and causes sodium reabsorption.
486 Chapter 9. Molecular enaocrinoiogy

Table 92 Classification of hormones by Qin logical functions

Metabolism of carbohydrate, lipid, amino acid insulin, glucagon, epinephrine, cortisol, somatotropin

Water and sodium balance aidosterone, antidiuretic hormone

Calcium and phosphate metabolism parathyroid hormone, calcitriol, calcitonin

Reproductive processes estradiol, testosterone, progesterone, gonadotropic

hormones

Synthesis and secretion ol endocrine gland pituitary tropic hormones, liberins. and hypothalamic
hormones statins

Metabolic changes in hormane-synbiesizing cells eicosanoids, histamine, secretin, gastrin,

somatostatin, cytokines

The biological effect of hormones is manifested through their interaction with,

target cell receptors (see Ch. 4.3). The binding of a hormone to a receptor leads to the
formation of a chemical signal inside the cell that causes a specific biological response,
for example, a velocity changes of synthesis of enzymes and other proteins or their
activity change. Effecting a target cell, the hormone causes a specific response. For
example, the thyroid gland is a specific target for thyrotropin, which increases the
numbcrofacinarcellsof the thyroid gland and the rate of thyroid hormone biosynthesis.
Glucagon activates lipolysis in adipocytes, stimulates glycogen mobilization and
gluconeogenesis in the liver. A characteristic feature of a target cell is the ability to
perceive information encoded in the chemical structure of a hormone.
The initial stage of hormone action on the target cell is the hormone interaction
with a cell receptor. Target cells distinguish the corresponding hormone from a variety
of other molecules due to the presence of an appropriate receptor on the target cell
with a specific hormone binding site.
The receptors for peptide hormones and epinephrine arc located on the cell
membrane surface and contain three various domains (Fig. 9.2).

Ligand-binding
domain

Extracellular side

Cell membrane

Cytoplasm

Cytoplasmic
domains (I to VII) domain

Fig. 92. Hepta helical receptors

9.1. Role of Hormones in Regulation of Metabolism and Functions 487

The extracellular domain of mem brane-associated receptor located in the

N-terminus of the polypeptide chain on the outerside of the cell membrane: it is often
highly glycosylated and provides recognition and binding of the hormone.
The second domain is transmembrane anchoring hydrophobic sequences. Some
transmembrane domains of surface receptors coupled with G-proteins consist of
seven tightly packed a-helical polypeptide sequences. In another type of receptors,
the transmembrane domain contains only one a-helical polypeptide chain (c.g.. both
P-sLibunits of insulin receptor c^B2; Ch. 4. Fig. 4.15). The third cytoplasmic domain
initiates intracellular signaling that matches the recognition and binding of the
hormone with a specific intracellular response. Intracellular signaling is mediated by
covalent modification and activation of intracellular signaling molecules (e.g., STAT
proteins) ortho generation of secondary' messengers. The cytoplasmic receptor site of
hormones such as insulin, epidermal growth factor and insulin-like growth factor-1 on
the inner side of the membrane has tyrosine kinase activity. The cytoplasmic sites
of growth hormone receptors, prolactin and cytokines do not exhibit tyrosine kinase
activity, and arc associated with other cytosolic protein kinases that phosphorylate and
activate the receptors.
Steroid and thyroid hormone receptors arc located inside the cells. For example;
intracellular receptors for glucocorticoids arc localized in the cytosol, receptors for
androgens, estrogens and thyroid hormones arc located in the cell nucleus. Steroid
and thyroid hormone receptors contain 3 major functional domains (Fig. 9.3).

Domains: Variable ONA Ligand

J r~i t----------------- 1 Glucocorticoid

I MI J Mineralocorticoid

[ — I"" Progestins

Estrogens

ran j Vitamin D

Thyroid hormone

Fig. 9.3. Ligand bin ns to noma in - C-terminal region (on the right). DMA binds to central nomain.
Proteins associated wim tne promoter region binds to variable domain (on tne left)

The C-terminal region of the receptor polypeptide chain contains a hormone

recognition and binding site. The central part of the receptor includes the DNA binding
domain containing two finger-1 ike projections from the molecule, each of which has a
globular shape and is complexed with zinc. This provides interaction with chromatin
by fitting into the major groove of a double helix, of DNA. The third domain, called
488 Chapter 9. Molecular eoaocrmoiogy

the variable region of the receptor, is near the N-tcrminusof the polypeptide chain, it
binds to other proteins, that arc complexed with their D NA-binding sequences in the
regulatory region, of the gene controlling transcription.
The concentration of receptors within the cell or on its surface and their affinity to
this hormone arc normally regulated in various ways, and can also change in diseases or
when using hormones or their agonists as medicines. For example, when p-adrcnergic
agonists arc exposed to cells for several minutes in response lo a new addition of the
agonist, activation of adcnytvl cyclase slops and the biological response disappears.
Such, a decrease in the sensitivity of the receptor to the hormone (desens itizalion)
may occur as a result of a change in the number of receptors by a down-regulation
mechanism. The hormone binds to the receptor, the hormone-receptor complex
enters the cell through endocytosis (internalizes), where some receptors undergo
proteolytic cleavage under the action of lysosomal enzymes, and some arc inactivated,
separating from other membrane components. This leads to a decrease in the number of
receptors on the plasma membrane. For example, in the case of insulin, glucagon and
catecholamines, this can happen within a few minutes or hours. When the hormone
concentration decreases, the receptors return lo the cell surface, and the sensitivity
to the hormone is restored. Receptor activity, j.e.. its affinity for the hormone, may
also change as a result of the covalent modification, mainly by phosphorylation. The
concentration of intracellular receptors can also be regulated by the mechanism of
induction and repression.
According to the location of receptors and the nature of the signal used to mediate
hormonal action within the cell (mechanism of signal transduction). hormones can
be. divided into 2 groups (Fig. 9.4). The hormones of the first group arc hydrophilic
and cannot diffuse throughout lipid bi layer of cell membrane, therefore, they interact,
with plasma membrane receptors (peptide hormones, epinephrine, eicosanoids and
cytokines). The hormones of the second group are hydrophobic, they penetrate in
target cellsand interact with intracellular receptors.
To deliver a hormone message to the cell the hormone (primary messenger) binds
to a receptor and causes a change in the receptor conformation. This change initiates
a sequence of biochemical events that include the conjugation of some molecules with
others (signal transduction). Thus, a signal is generated that regulates the cellular
response by changing activity or amount of enzymes and other proteins (Fig. 9.5).
Depending on. the mechanism of hormonal signal transduction to the cells, the rale of
metabolic reactions varies:
► as a result of changes in the activity of enzymes;
► as a result, ofchanges in. the amount of enzymes.
The synthesis of hormones can occur cither in specific cells of specialized
endocrine glands, or in cells that perform additional functions. Many protein
hormones, such as growth hormone (GH). PTH, prolactin, insulin and glucagon,
arc produced in specialized cells using standard protein synthesis mechanisms
common to all cells. These secretory cells contain secretory granules to store large
amounts of the hormone and release the hormone in response to certain signals.
9.1. Role of Hormones in Regulation of Metabolism and Functions 489

Peptide or amine hormone Steroid or Hnyroid hormone

binds la receptor on the enters tne celt; hormone-

pre-ejchfing newly Eynmesized

enzyme proteins

Fig. 9.4.Two general mechanisms of hormone action The peptide and amine hormones act throughout
surface receptors and steroid and thyroid normones act throughout intracellular receptors

Steroid hormones arc derived from cholesterol that is modified by various

hydroxyiations, methyiadon and demethylation reactions to form glucocorticoids,
androgens, and estrogens and their biologically active derivatives. Peptide hormones
(e.g., insulin and glucagon, somatostatin, the parathyroid hormone, calcitonin,
the hypothalamus and pituitary gland hormones) arc synthesized on ribosomes in
the form of longer precursor proteins (prohormon.es). Then they arc packaged into
secretory vesicles and protcolytically cleaved to form the active peptides which may
have 3 io 200 or more amino acid residues.
490 Chapter 9. Molecular enaocrinoiogy

Fig. 9.5. The main stages of the hortooitai signal transduction to target cells

92. STRUCTURE, SYNTHESIS AND BASIC ACTION OF HORMONES

OF THE HYPOTHALAMUS AND PITUITARY GLAND
The hypoibaJamus is a key mediator in the hierarchical system combining the
higher centers of the central nervous system and endocrine glands. Two groups of
peptide hormones arc synthesized in the cells of the hypothalamic neurons. First
group hormones through the system of hypothalamic-pituitary vessels enter the
anterior pituitary, where they stimulate or inhibit the synthesis of tropic hormones.
Second group hormones such as oxytocin and vasopressin, enter into the posterior
pituitary gland through the axons of nerve cells store into vesicles and secrete into
blood as a response to the appropriate signals (Table 93).
9.2. Structure, Synthesis and Basic Action of Hormones of tne Hypothalamus and Pituitary Gland 491

Table 9.3, Biological functions of principal hypothalamic hormones

Thyrotropin-re leasing hormone (TRH) Stimulates Thyrotropin and prolactin secretion

Corticotropin-releasing hormone (CRH) Stimulates Corticotropin secretion
Gonadotropin-releasing hormone (GnRH) Stimulates LH and FSH secretion
Growth hormone-releasinq hormone (GHRH) Stimulates GH secretion
Somatostatin (SS) Inhibits GH secretion
Prolactin-releasing factor Stimulates prolactin secretion
Prolacto statin Inhibits prolactin secretion

Thyrotropin-releasing hormone (TRH) is a tripcptidc; the precursor of human

TRH synthesis occurs in various parts of the hypothalamus, but to a greater extent
in the paraventricular nucleus, as well as in other areas of the central nervous system
where it acts as a neurotransmitter to increase motor activity and blood pressure. The
active hormone formation occurs by partial proteolysis. In the anterior pituitary gland,
TRH stimulates the synthesis and secretion of thyrotropin, and has a stimulating effect
on the synthesis of many other hormones. As a result of the thyroliberin interaction
with the receptors of the pituitary cell plasma membrane, intracellular cAMP, and
Ca2 concentrations increase. Membrane receptors of TRH transduce signals through
adenylyl cyclase and phosphatidylinositol systems. Specific proteases inactivate the
hormone in target cells and in blood. The half-life (a biological half-life, denoted as
Tl/2, is the time it takes to lose half of a physiologic hormone activity or may also
describe the time it takes for the blood plasma concentration of a hormone to halve
(plasma half-life) its steady-state) of TRH in the blood is 3-4 minutes.
Corticotropin-releasing hormone is a polypeptide and, like other peptide hormones,
it is synthesized as a prohormonc. The hormone Tl/2 in blood plasma is 60 min.
It is mainly formed in the hypothalamus, however, also found in other parts of the
central nervous system, where it acts as a mediator, participating in response to various
stressful situations. In the anterior pituitary gland, the corticotropin-releasing hormone
increases the synthesis and secretion of proopiomelanocortin and the formation of
corticotropi n. The hormone receptors arc located in the plasma membrane of cells
as part of the adenylyl cyclase complex. Stimulation of ACTH secretion requires the
presence of Ca2 ions. An increase in intracellular calcium is likely to result from
phosphorylation of calcium channel proteins.
Gonadotropin-releasing hormone is a dccapcptide. It stimulates the synthesis and
secretion of 2 pituitary hormones - LH and FSH. Besides hypothalamus, neurons
with GnRH arc also found in other areas of the central nervous system that control
emotional and sexual behavior. The GnRH membrane receptor is part of the inositol
phosphate complex. Its activation stimulates protein phosphorylation and the
Ca2 mobilization, that leads to the hormones release. The half-life of gonadotropin­
releasing hormone in the blood is approximately 5-7 minutes. Specific proteases
inactivate GnRH.
Growth hormone-releasing hormone, a polypeptide, in the anterior pituitary gland
stimulates the synthesis and secretion of growth hormone. Signal transduction is
492 Chapter 9. Molecular eoaocrmoiogy

accompanied by an increase in the concentration of both cAMP and calcium ions.

The half-life ofgrowth hormone-releasing hormone in the blood is around 7 minutes.
GHRH is used in clinical practice to diagnose pituitary dysfunction.
Somatostatin was primarily isolated from the hypothalamus, but later it turned out
that it is synthesized in many cells located outside the hypothalamus: in the stomach,
intestines, pancreas, in the region of peripheral nerve endings, in the placenta, adrenal
glands and in the retina. Somatostatin acts as a hormone and mediator, causing
inhibition of the secretory processes, reducing the activity of smooth muscles and
neurons. Like other peptide hormones, somatostatin interacts with the receptors of
the plasma membrane of cells. There arc 5 types of somatostatin receptors associated
with G-proteins. Ah types of receptors arc expressed in the anterior pituitary and
hypothalamus and have varying degrees of affinity for different structural forms
of somatostatin. Somatostatin receptors are present in many hormone-secreting
tumor cells. This circumstance is used to develop methods for the early diagnosis of
pancreatic tumors, pheochromocytoma, thyroid cancer, kidney and breast cancer.
The result of somatostatin signal transduction is a decrease in the level of intracellular
concentration of cAMP and Ca2 in the cell cytosol. Somatostatin inhibits the
secretion of many molecules, such as growth hormone, glucagon, insulin, gastrin,
secretin, vasoactive intestinal peptide (VIP), cholecystokinin, calcitonin, parathyroid
hormone, immunoglobulins, and renin; it also inhibits the secretion of bicarbonatcs
and pancreatic enzymes, reduces blood flow in the digestive tract, and reduces the
bile secretion.
The pituitary gland secretes a large number of hormones involved in. the regulation
of various biochemical processes and physiological functions. In the anterior lobe
of the pituitary gland, the so-called tropic hormones arc synthesized that stimulate
synthesis and secretion of hormones of other endocrine glands or affect the metabolic
reactions in other target tissues (Table 9.4). The posterior pituitary gland secretes
hormones that regulate mainly water balance and lactation.
The pituitary’ hormones secretion is due to a combination of nerve and humoral
signals, and the same agonist (for example, norepinephrine) can cause opposite
changes in the pituitary hormones secretion. On the other hand, the secretion of each
hormone can be controlled by numerous factors.
The synthesis and secretion the anterior pituitary hormones arc regulated by
the hypothalamic hormones, that enter the pituitary through the portal system of
blood vessels connecting the hypothalamus and the anterior pituitary. Moreover,
the secretion of hypothalamic and pituitary gland: hormones is feedback regulated by
hormones produced in corresponding target tissues.
Most of the anterior pituitary hormones arc glycoproteins, some of them arc
peptides. According to the mechanism of synthesis and biological functions,
these hormones can be divided into 3 groups. The first group involves prolactin
and GH. Thyrotropin. Luteinizing hormone and Follicle-stimulating hormone
arc included in the second one. The third group contains hormones formed from
p roop iom clanocort i n.
9.2. Structure, Synthesis and Basic Action of Hormones of tne Hypothalamus and Pituitary Gland 493

ladle 9.4. Biological 1uisclions of principal hormones of tde anterior pituitary gland

Growth hormone (Somatotropin) Stimulates postnatal growth of ihe skeleton and

soft tissue; promotes growth in stature and mass;
stimulates the production of insulin-Iike growth
factor (fGF-l)z stimulates protein synthesis: usually
inhibits glucose utilization and promotes fat
utilization

Thyra id-stim ulati ng ho rmo ne (TS H), o r Thy retro pi n Stimu I at e s synthesis and se cretio n of thyro id
or thyrotropic hormone) hormones

Prolactin (PR L. luteotrop ic hormon e o r Iuteotropin j Stimu lates m ilk secretio n an d mammary g rawth

Luteinizing hormone (LH) In women, ovulation of ripe Io Ilk le and formation

of corpus luteum; stimulates estrogen and
progesterone synthesis by corpus luteum. in men,
induces androgens synthesis tn Leydig cells

Follicle-stimulating hormone (FSH) In women, it stimulates the growth of fa Hides

and estrogen secretion. In men. stimulates
spermatogenesis

Adrenocorticotropic hormone (ACTH. Stimulates adrenal growth, and corticosteroid

adrenocorticotropin or corticotropin) synthesis and secretion

P-Ljpotropin ffl-LPH) Stimulates lypolisis

Prolactin is synthesized in lac tot rop hie cells of the anterior pituitary as a
prohormonc. The number of J actol rop hie cells increases sharply during pregnancy
under estrogens action. Prolactin receptors arc present in many tissues: in the liver,
kidneys, adrenal glands, testes, ovaries, uterus, and other tissues. The prolactin main
physiological function is the stimulation of lactation. Prolactin induces the synthesis
of a-lac tai bum in and casein, activates the synthesis of phospholipids and TAG.
Prolactin, affects growth processes to a much lesser extent than growth hormone. In
men, prolactin increases the sensitivity of Leydig cells to luteinizing hormone, thus
maintaining the necessary level of testosterone synthesis; in the kidneys, prolactin
reduces the water excretion, affects the reabsorption of Na* and K ions; prolactin also
enhances humoral and cellular immunity. The synthesis and secretion of prolactin
is stimulated by thyrotropin-releasing hormone; serotonin, oxytocin, acetylcholine,
and dopamine have an inhibitory effect. Like most hormones, prolactin is secreted
into the blood occasionally al intervals of 30-90 minutes. The maximum secretion is
observed 6-8 hours a her a falling asleep. I n women the blood prolactin concentration
is 8—10 ng/ml, and in men that is 5-8 ng/ml. The half-life of prolactin in scrum is
15—20 minutes. The placenta produces a hormone (placental lactogen), homologous
in amino acid composition to growth hormone and prolactin. All 3 hormones have
common antigenic determinants and have growth-stimulating and lactogenic activity.
There is a hypothesis that the genes of these hormones arose as a result of the
duplication of a single precursor gene.
494 Chapter 9. Molecular eoaocrmoiogy

Somatotropin, GH. is synthesized as a pro hormone in somatotrophic cells, which

arc the most numerous in the anterior lobe of the pituitary gland. GH is secreted
episodically from the pituitary gland with a major surge al the onset of slow-wave sleep
and less conspicuous secretory episodes a few hours after meals. Under the influence
of various stimuli (exercise, fasting, protein foods and arginine amino acid), GH
secretion.is amplified., whereas an excess of fuels such as glucose and lipid intermediates
inhibits GH release in. healthy adults. Pulsatile GH secretion is influenced by a number
of neurogenic, metabolic, and hormonal factors. GRH controls GH synthesis by
stimulating transcription of GH mRNA. while somatostatin has the main inhibitory
effect and determines the liming and amplitude of GH pulses.
Growth hormone receptors arc located in the plasma membrane of liver cells,
adipose tissue, testicles, corpus luteum. skeletal muscle, cartilage, brain, lungs,
pancreas, intestines, heart, kidneys and lymphocytes. GH is found to bind to the
dimerized receptor and causes an internal rotation of the receptor, activating the
rece ptor com plex. Ty rosi no k i nascs o f t he J a n u s fa m i ly (J A K2 p rotci n s) assoc iated wit h
each monomer of the GH-R dimer contain tyrosine residues in the kinase domain.
These residues are cross-phosphorylated, followed by the activation of Janus kinases
and phosphorylation of the dimeric GH-R intracellular domain that initiates different
intracellular signaling pathways. The transcription of multiple genes is affected as a
result of the activation of these patliways.
The main action of growth hormone is to regulate the metabolism of proteins and
processes associated with body growlhand development. The metabolic effects of G H
associated with the uptake and oxidation of fuels manifests in adipose tissue, muscle,
and liver and can. be both as a direct response to growih hormone action and as a
result of GH actions mediated by molecules that arc synthesized in response to the
growth hormone interaction with the plasma membrane receptors of various tissues,
mainly the liver, and called somatomedins. These molecules arc highly homologous
to each other, as well as to proinsulin, and have insulin-like activity and potent
growth-promoting action, so they arc called insulin-like growth factors (IGF-l, or
somatomedin C; 1GF-2, or somatomedin A). IGF-l is a single-chain polypeptide
with basic properties, and 1GF-2 is a polypeptide with acidic properties. In the
blood, approximately 95% of somatomedins circulate bound with proteins. The IGF-
I synthesis is more dependent on the blood growth hormone concentration than the
IGF-2 synthesis. At the same lime, the synthesis and secretion of growth hormone as
feedback inhibited by IGF-L
Insulin-1 ike growih factors exert their effects in various wavs: endocrine, paracrine
and autocrine. Like the insulin receptor, the IGF-l receptor has tyrosine kinase
activity and initiates a cascade of phosphorylation reactions of other proteins involved
in various intracellular processes, including activation of gene transcription. In most
eases, IGF-l, like insulin, initiates cell development, but al much lower, almost
physiological concentrations. This indicates that insulin-like growth factors arc more
active in relation to their effect on cell growth and development.
Growth hormone increases the amino acid transport into muscle cells; stimulates
protein synthesis in bones, cartilage, muscles, liver and other internal organs, thereby.
9.2. Structure, Synthesis and Basic Action of Hormones of trie Hypothalamus and Pituitary Gland 495

enlarging the width and thickness of bones, and the growth of other tissues, including
connective tissue, muscles and internal organs; enhances the total amount of R.NA,
DNA and the total number of celts. On the whole, the overall effect of G H on protein
turnover is an anabolic one.
In. adipose tissue, GH increases lipolysis leading to increased circulating free fatly
acids. In muscles, these fatty acids are preferentially served as fuels, and glucose uptake
is indirectly suppressed! with glycolytic pathway decreased. In liver, the GH stimulates
production and release of ICF-I. accelerates hepatic gluconeogenesis, while it has
modest effects on. liver glucose uptake, utilization, or storage. Il also enhances the
oxidation of fatty acids in the liver and consequently stimulates ketogenesis with
increased blood ketone body level in fasting (when insulin level is tow).
Thus, GH exerts diverse effects on tissues. Many of the physiological effects of
GH arc still unknown. Now targets ofGHR signaling arc steadily emerging, and the
metabolic actions of GH may not be as clear as was initially believed. Understanding
the role of GH in physiological and pathological states could contribute to the
development of new therapeutic strategics.
Thyrotropin, LH, and FSH arc glycoproteins. Thyrotropin (TSH) is synthesized
in thyrotrophic cells of the anterior pituitary' gland. The thyrotropin secretion is
stimulated by thyrotropin-releasing hormone, and is mainly inhibited by the thyroid
hormones level increase. The peak TSH secretion is observed in the hours immediately
preceding sleep, followed by a decrease during the night.
The main biological function of thyrotropin is to stimulate the synthesis and
secretion of the thyroid hormones, thyroxine (T4), and Tr from the epithelial cells
making up the follicles of the thyroid gland. Thyrotropin binding with plasma
membrane receptors activates adenylyl cyclase. Thyrotropin has two types of effects
on the thyroid gland. First effects manifest rapidly (within a few minutes) and i nclude
stimulation of the thyroid hormone's synthesis and secretion. The second group of
effects that has several days exhibited includes stimulation of the synthesis of proteins,
phospholipids and nucleic acids and an. increase in the size and number of thyroid
cells. A numberofimmunoglobulinsG, interacting with thyrotropin receptors, mimic
the effects of the hormone. Similar immunoglobulins arc found in most patients with
hyperthyroidism.
The group of hormones related to glycoproteins also includes the gonadotropic
hormones of the pituitary gland of LH and FSH and the placental chorionic
gonadotropin (hCG). FSH and LH play the important biological roles in the
reproductive system in mates and females. Also, hCG, which is initially produced by
the human trophoblast upon implantation in the uterus, mimics the actions of LH and
is essential for maintenance of pregnancy in its early stages.
Frequently several active hormones can be carved out of the same prohormonc.
Macromolecule POMC (proopiomelanocortin) is an example of multiple hormones
encoded by a single gene. The POMC gene encodes a large polypeptide that is
progressively cleaved into at least nine biologically active pepLidcs (Fig. 9.6). POMC
is synthesized in the anterior and intermediate lobes of the pituitary and in some
other tissues (intestines, placenta). The polypeptide chain consists of 265 amino acid
496 Chapter 9. Molecular endocrmoiogy

residues. The processing of this large precursor molecule in different cells proceed
differently with the formation of a different set of peptides. After removal of the
N-terminal signal peptide, the polypeptide chain is split into two fragments: ACTH
(39 a.L)and [3-lipotropin (42-134a.k.). Additional proteolysis leads to the formation
ofa-and p-MSH (melanocyte-stimulating hormone), endorphins, corticotropin-like
hormone intermediate lobe peptide (CLIP} and y-lipotropin. P-MSH and CLIP of
the intermediate fraction in humans arc practically not formed, since in. adults the
intermediate fraction is not developed. B-lipotropin, y-lipotropin and p-endorphin
were found in the human pituitary gland. The function of all products of the POMC
proteolysis is not sufficiently studied.

Fig. 9.6. Processing of proopiomelanocortin (POMC). The protein POMC is produced Dy the anterior
pituitary in response to corticotropin releasing hormone (CRH). The N-terminai region is removed,
and the remainder is cleaved to form adrenocorticotropic hormone (ACTH) and 0-lipotropin (B-LPH).
Additional cleavages produce the melanocyte stimulating hormones (a- and P-MSH), endorphins,
corticotropin-like intermediate lobe peptide (CLIP), and y-fipotropin fr-LPH)

Corticotropin (ACTH) is a peptide hormone synthesized in the cells of the

anterior pituitary gland by the corticotropin-releasing hormone stimulation.
.ACTH secretion is pulsatile and exhibits a characteristic circadian rhythm, peaking
at 6 a.m. ACTH levels arc increased by trauma, bums, surgery, intoxication with
chemicals, bleeding, pain, psychological stress, exercise. The hormone life-lime
in the blood is 15-25 minutes. The mechanism of ACTH action includes the
interaction with the reccpiorofthe plasma membrane, activation ofadcnylvl cyclase,
and phosphorylal ion of proteins involved in the synthesis of corticosteroids. These
effects arc enhanced in the presence of Ca2' ions. In cells of the adrenal cortex,
ACTH stimulates the hydrolyzis of cholesterol esters and increases the intake of
cholesterol in LOL; it also stimulates the cholesterol conversion to pregnenolone
and induces the synthesis of mitochondrial and microsomal enzymes involved in
the corticosteroids synthesis.
9.3. Structure, Syntnesis and Basic Action of Principal Hormones Regulating Fuel Metabolism 497

In the human, the posterior pituitary gland stores and secretes two important
nonapepiides, oxytocin and a nt id in retie hormone, into the circulation. These hormones
arc synthesized in hypothalamic neuronal cell bodies, together with a ncurophysin
iliat accompanies the hormone from the cell body through the axon to the nerve
ending awaiting an appropriate signal to release the complex into the bloodstream.
Oxytocin stimulates smooth muscle contraction in the uterus and bieast; it functions
in parturition (childbirth) and lactation. Antidiuretic hormone (ADH), also known as
vasopressin (VP), controls water balance (this hormone is covered in Ch. 9.9}.

93. STRUCTURE, SYNTHESIS AND BASIC ACTION OF PRINCIPAL

HORMONES REGULATING FUEL METABOLISM
Insulin is a polypeptide consisting of two polypeptide chains. Chain A contains
21 aminoacid residues; chain B has 30 amino acid residues. Both, chains arc joined by
two disulfide bridges. The insulin A-chain contains an intramolcculardi.sulfide bridge
(in Fig. 9.7).
Insulin biosynthesis begins with the formation of inactiveprecursors, prcproinsulin
and proinsulin, which arc transformed into an active hormone as a result of sequential
proteolysis. Biosynthesis of prcproinsulin begins with the formation of a signal
peptide on polyribosomes associated with the endoplasmic reticulum in B-ccIlsofthe
pancreas. The signal peptide penetrates the lumen of the endoplasmic reticulum and
sends a growing polypeptide chain to the ER. After the prcproinsulin synthesis has
completed, the signal peptide is cleaved ofT (Fig. 9.8).
Prepfoifisulii'i Proi/isulifl Mature insulin

Fig. 9X Structure of insulin. Mature insulin is farmed from pre proinsulin Dy proteolytic processing.
Removal of tne signal sequence ano formation of three disulfide Dorias produces proinsulin. Removal
of the C peptide from prornsulin produces mature insulin, composed of A and B chains
498 Chapter 9. Molecular enaocrinoiogy

Fig. 9.8. insulin Biosynthesis scheme in p-cells of Langerhans islets. ER — endoplasmic reticulum.
1 — formation of the signal peptide; 2 — synthesis of preproinsulin; 3 — cleavage of me signal
peptide; 4 — transport of proinsulin lo Ihe Golgi apparatus; 5 — the conversion of proinsulin to
insulin and C-peptide and tne insulin and C-peptioe incorporation in secretary granules; 6 — insulin
and C-peptide secretion

Proinsulin (86 amino acid residues) enters the (iolei apparatus, where it is
cleaved by specific proteases al several silos lo form insulin (51 amino acid residues)
and C-peptide consisting of 31 amino acid residues. Equimolar amounts of insulin
and C-peptide arc incorporated in secretory granules of the pancreatic beta cells. In
granules, insulin combines with zinc to form d imers and hexamers. The kidney is the
principal site of C-peptidc degradation. Because little degradation of the C-pc pt ide
occurs in the liver, its concentration in blood is useful for estimating the rate of
insulin secretion and evaluation of beta-cell function.
The secretion of insulin is primarily regulated by blood glucose concentration.
The GLLFT-2 proteins on the membrane of pancreatic p-cclls have a relatively high.
Km for glucose (- 20 mM) and, therefore, provide an active glucose transport from
the blood into the p-cctls of the pancreas. Entering the pancreatic cells, glucose is
phosphorylated and further oxidi zed. This causes an increase in the ATP/ADP ratio in
the cell, which initiates closure of the ATP-dependent K1 channels. This depolarization
causes voltage-gated Ca2' channels to open, initiating docking and secretion of insulin
from secretory vesicles. After secretion into the blood, insulin oligomers disintegrate.
9.3. Structure, Synthesis and Basic Action of Principal Hormones Regulating Fuel Metabolism 499

The half-life of insulin in blood plasma is 3 -10 minutes, C-peptide is about 30 minutes.
Insulin is inactivated by the enzyme insulinase, mainly in the liver and to a lesser extent
in the kidneys. The main stimulus of insulin synthesis and secretion is glucose. Insulin
secretion is also enhanced by sonic amino acids (especially arginine and lysine),
ketone bodies and fatty acids. Epinephrine, somatostatin, and some gastrointestinal
peptides inhibit insulin secretion.
Insulin is the main anabolic hormone. It participates in the regulation of
metabolism, transport of glucose, amino acids and ions, and also protein synthesis.
Insulin affects the processes of replication and transcription, thus participating
in the regulation of cell differentiation, proliferation and cell transformation. The
participation of insulin in the regulation of metabolism is discussed in. the relevant
units (see also Ch. 4.3 and Pig. 4.12). The effect of insulin on key metabolic enzymes
is presented in Tabic. 9.5.

Table 9.5, The insulin effect on key metabolic enzymes

Activation

Phosphodiesterase Phosphodiesterase LP-lipase

Phosp holructoki nase-2 Pyruvate kinase Pyruvate kinase

Pyruvate kinase Pyruvate dehydrogenase Acetyt-CoA carboxylase

Pyruvate dehydrogenase

Glycogen synthase Glycogen synthase

Glycogen phosphorylase Glycogen phosphorylase

Phosphorylase kinase

Acetyt-CoA carboxylase

HMG-CoA-reductase

Induction

Glucokinase Fatty acid synthase

Citrate lyase

Fatty acid synthase

Malic enzyme

Pyruvate kinase

AcelyFCoA carboxylase

Phosp hofructoki nase-1

Glucose-6-phosp hate de hydrogenase

Repression

Fructose bisphosphatase

Phosp hoenolpyruvate carboxykinase

500 Chapter 9. Molecular eoaocrmoiogy

Insulin stimulates the glucose transport into insulin-dependent tissues, muscle

cells and adipose tissue, by promoting the recruitment of glucose transporters (GLUT-
4) to the cell membrane. In liver cells, insulin induces the glucokinase synthesis.
Glucokinase irreversibly converts free glucose to glucose 6-phospatc thereby keeping
low free glucose concentration in the cells, wrhich contributes to glucose transport
from blood along the concentration gradient.
Insulin stimulates the utilization of glucose in cells in different ways. .About 5058 of
glucose is oxidized in glycolytic pathway, 30-40% is converted to fat and about 10% is
accumulated as glycogen. The overall result of stimulating those processes isa decrease
in blood glucose concentration.
In liver, insulin increases the activity and the amount of key glycolytic enzymes:
glucokinase, phosphofructokinasc-1., pyruvate kinase. In the liver and muscles,
insulin decreases the c AM P concentration as a result of phosphodiesterase activation.
In addition, insulin activates phosphatases that dephosphorylate and activate glycogen
synthase increasing glycogen synthesis and, inhibiting its breakdow n.
The effects of insulin due to phosphorylation and dcphosphorylation of enzymes
occur rapidly, within a few seconds and minutes. Insulin inhibits gluconeogenesis by
repressing the synthesis of the key gluconeogenesis enzymes, phosphocnolpyruvatc
carboxy kinase and fructose bisphosphatase.
In the liver and adipose tissue, insulin promotes the TAG synthesis, providing
this process with the substrates (acctyl-CoA. glycerol-3-phosphate and NAD PH)
generated from glucose. In adipose tissue, insulin activates acetyl-CoA carboxylase
and LP lipase and induces the synthesis of fatly acid synthase. acctvI-CoA carboxylases
and LP lipases. It also activates phosphatase, which in turn dephosphoryiates and
thereby inactivates the hormone-sensitive TAG lipase with being inhibited the TAGs
mobilization from adipocytes. Thus, insulin decreases concentration of free fatty acids
circulating in the blood.
Insulin promotes the consumption of neutral amino acids in muscles and the
protein synthesis in liver, muscles and heart: it stimulates the cell proliferation in
tissues, and is also likely to be involved in in vivo growth regulation.
Glucagon is a single-chain polypeptide consisting of 29 amino acid residues.
Glucagon biosynthesis occurs in thca-cclls of the islets of Langerhans. Inactive larger
precursor, preproglucagon, produced on the RER enters the lumen and sequentially
converts lo proglucagon and glucagon by partial proteolysis. Glucose and insulin
inhibit the secretion of glucagon; such molecules as amino acids, fatty acids and
neurotransmitters (c.g., epinephrine), stimulate the glucagon secretion. The half­
life of the hormone is about 5 minutes. In the liver, glucagon is rapidly degraded by
specific proteases.
The effects of glucagon arc basically the opposite of the effects of insulin. The
main target cells of glucagon are the liver and adipose tissue. By binding to receptors
on the plasma membrane of target cells, glucagon increases the cAM. P level in cells. I n
hepatocytes, this leads to the activation of glycogen phosphorylase and to a decrease
in glycogen synthase activity with glycogen mobilization accelerated. Phosphorylation
of pyruvate kinase and BEE causes inhibition of glycolysis and acceleration of
9.3. Structure, Synthesis and Basic Action of Principal Hormones Regulating Fuel Metabolism 501

gluconeogenesis- In addition, glucagon stimulates gluconeogenesis, inducing the

synthesis of enzymes: glucose-6-phosphatase, phosphocnolpyruvate carboxykinasc,
fructose -1,6-bis-phosphalasc. In adipose tissue cells, glucagon activates the hormone­
sensitive TAG lipase through the adenylyl cyclase cascade and stimulates lipolysis.
Thus, i n contrast to insulin, glucagon stimulates the mobilization of the main energy
sources — carbohydrates and fats.
Thyroid hormones are synthesized in the composition of the protein — thyroglobulin
(Th) (Fig. 9.91.

Tc£] witfiTjr Tgo wiifi DIT Tgb wiih T.«

Residues

H2O2

Colloid OH

Stood lexfire pump

Ta I*

Fig. 9.9, Synthesis of the tftyroid hormones (I3 and IJ. Synthesis of tn e protein thyroglobulin (Tgb) in
thyroid follicular cells. Active transport of iodide by the sodrum/iodide symporter into the thyroid gland
follicular ceils. Oxidation of iodide by peroxidase, s 0 di nation cf Tyrosyl residues within and formation of
monoiodotyrosyl (MiT) and diiodotyrosyl (DlTj within the protein thyroglobulin. Transfer and coupling
of iodotyrosines within thyroglobulin to form thyroxine (Tj and triiodothyronine {Tj. Storage of
thyroglobulin as the colloid in the lumen of The thyroid follicle. Endocytosis of the colloid back into
the thyroid epithelial cell. Proteolysis of thyroglobulin with concomitant release of T+ and I3 as well
as free and io dothyronines. Secretion of T# and T3 into the blood. De iodination of iodotyrosines within
the thyroid follicular cells for reutil izati on of the liberated iodine. R£R — rough endoplasmic reticulum

Thyroglobulin needed for the synthesis of thyroid hormones is a large glycoprotein

composed of 1.15 tyrosine residues. It is the matrix for thyroid hormone synthesis and
502 Chapter 9. Molecular enaocrinoiogy

the form of hormone storage in the gland. Thyroglobulin is synthesized on ribosomes,

glycosylated in the cistcrnae of the endoplasmic reticulum, translocated to the Golgi
apparatus, and packaged in secretory vesicles, which discharge it from the apical
surface into the lumen.
Iodide entering at the basolateral surfaces of the follicular cell diffuses throughout
the follicular cell and exits from the apical membrane by a sodium-independent
iodide transporter. Then the thyroid peroxidase located al the apical border of the
thyroid acinar cell converts iodide to higher oxidized state (F, iodinium ion) in the
presence of hydrogen peroxide. Oxidized iodine reacts with tyrosine residues to form
monoiodoihyronincs (MIT) and diiodothyronincs (DIT), then two DIT molecules
condense to form Tr and MIT and DIT to form Tr lodithiroglobulin is transported
into the cell by endocytosis and is hydrolyzed by lysosome enzymes with T5 and
T4 releasing.
T1 is the main biologically active thyroid hormone: its affinity for the target cell
receptor is 10 limes higher than that of T4. In the peripheral tissues deiodination of
the fifth carbon atom of Tt may result in formation of a so-called ^reverse* form
Ts which is almost biologically inactive. In blood, the iodoihyronines are complexed
with a thyroxine binding protein. Just 0.03% T4 and 0.3% T3 are in. a free state that is
a biologically active form ofiodothyronin.es. Transport proteins serve as the hormone
storage in the blood providing an additional amount of free hormones. Synthesis and
secretion of thyroid hormones arc regulated by the hypothalamic-pituitary system
(Fig. 9.10).
Thyroid hormone target cells have 2 types of receptors. Some receptors mediate
major effects. They arc located in the nucleus and constantly bound with hormones
for interacting with certain DNA sequencesand participating in the gene expression
regulation. Other receptors arc located in the cytosol of the cells. They have a tower
affinity for thyroid hormone and arc likely to provide hormone binding to keep
them in close proximity to the cell nucleus and prevent their escape back into the
plasma.
In physiological concentrations thyroid hormones accelerate protein synthesis,
stimulate growth processes and cell differentiation. In this view; they arc synergists of
growth hormone, in addition. accelerates the transcription of the growth hormone
gene. In animals with Tn deficiency, pituitary cells lose their ability lo synthesize
growth hormone. In. high concentrations T3 inhibits protein synthesis and stimulates
catabolic processes and a negative nitrogen balance is an indicator of this.
In different cells, Ttstimulates the Na ’. K ’ -ATPasc, which, consumes a significant
part of the energy utilized by the cell. In the liver, thyroid hormones accelerate,
glycolysis, cholesterol synthesis, and bile acid synthesis. In the liver a nd adipose tissue,
T5 increases the sensitivity of cells to the epinephrine action and indirectly stimulates
lipolysis in adipose tissue and glycogen mobilization in the liver. In physiological
concentrations, T, increases glucose capture by muscles, stimulates pro Lein synthesis
and grows of muscle. The hormone also promotes glycolysis increasing the sensitivity
of muscle cells to epinephrine.
9.3. Structure, Synthesis and Basic Action of Principal Hormones Regulating Fuel Metabolism 503

Hypothalamus

Pituitary

Thyroid

Fig. 9.10. Feedback regulation of tnyroid hormone secretion. TRH stimulates (+) the release of TSH,
wnicn stimulates {+) tne release of T3 and Tr T4 is converted to T, in the liver and other cells. T3 and
T( inhibit (-) the release of TSH and of TRH

Thyroid hormone also participates in the formation of a response to cooling by

increasing heat production, stimulating the norepinephrine secretion, and increasing
the sensitivity of diesympathetic nervous system to norepinephrine. Norepinephrine,
released from sympathetic terminals and acting via P-adrenoceptors and cAMP.
stimulates both thermogenin synthesis and activity. Thcrmogenin, an uncoupling
protein + is an inner-membrane mitochondrial protein in brown adipocytes in
mammals (though normal adults have very low residual brown fat}. This protein
functions as a proton transporter and allows the dissipation as heat of the proton
gradient generated by the respiratory chain and thereby partially uncoupling oxidative
phosphorylation and! increases heal generation. Norepinephrine also increases the
permeability of brown adipose tissue and skeletal muscle to Na\To maintain Na and
K' intracellular balance, Na', K -ATP'asc transports Na' out of the cell in exchange
for K*. The increased hydrolysis of ATP by Na', K:-ATPasc stimulates the fuels
oxidation and the regeneration of more ATP and heat from oxidative phosphorylation.
Over a longer time-course, thyroid hormone increases the level of Na , K‘-ATRase
and many of the enzymes of fuel oxidation , and increases oxygen absorption bv cells
in most tissues.
Steroid hormones in mam malian can. be divided into several groups:
► estrogens and progestins (female sex steroids);
» androgens (male sex steroids);
504 Chapter 9. Molecular eoaocrmoiogy

► mineralocorticoids (aldosterone);
► glucocorticoids (cortisol);
► eaten riol (active form of vitamin Ds).
In (he adrenal cortex that is anatomically divided into three zones the three
di fie rem major classes of the steroid hormones, or corticosteroids, arc produced:
mineralocorticoids arc formed in zona glomcrulosa: glucocorticoids are synthesized
in the zona fasciculate; and androgens arc formed in zona reticularis. Glucoconicoids,
C,, steroids, play an important rote in adaptation to stress. They have a variety of
effects, and the most important is the stimulation of gluconeogenesis. The main
human glucocorticoid is cortisol. Mineralocorticoids, steroids, are required to
maintain Na* and KJ levels in living cells. The most active this group hormone is
aldosterone. Androgens arc C|9 steroids. The adrenal cortex generates androgen
precursors, the most active is dehydroepiandrosterone (IJHEA) and the weakest
is androstenedione. The more potent androgen testosterone is synthesized in the
adrenal glands in a small amount. The adrenal androgens are converted into more
active androgens outside the adrenal gland, in the adrenal glands, trace amounts
of testosterone can be converted to estradiol. Normally, the adrenal production of
Cl9steroids playsan insignificant role.
A com mon precursor ofal I corticosteroids is cholesterol. The sources of cholesterol
for the corticosteroids synthesis arc cholesterol esters that enter the cell as part of LDL
or deposited in. the cell. The release of cholesterol from its esters and the synthesis of
corticosteroids arc stimulated by corticotropin. More than 40 metabolites with different
structure and biological activity arc formed in corticosteroid pathway. The final
product depends on the set of enzymes in the cell and the sequence of hydroxylation
reactions (Fig. 9.11). The main corticosteroids with pronounced hormonal activity arc
cortisol, aldosterone and androgens.
At the first stage ofcorticostc.ro id synthesis cholesterol is converted to pregnenolone
by cleaving the 6-carbon fragment from the cholesterol side-chain and oxidizing the
carbon atom Cjn. Then pregnenolone can be converted both io progesterone (C3I.)T
the precursor of cortisol and aldosterone, and to CN steroids, androgen precursors.
The primary hydroxylation of progesterone by 17-hydroxylase, and then 21- and
11-hydroxylase leads to the synthesis of cortisol. Reactions of aldosterone formation
include hydroxylation of progesterone, first with 21-hydroxylase, and then with
11-hydroxylase. The final steroid product is dependent on the set of enzymes in the
cell and the sequence of hydroxylation reactions.
Cortisol synthesis reactions occur in different compartments of the adrenal cortex
cells (Fig. 9.12).
The cortisol synthesis begins with the conversion of pregnenolone to progesterone.
This reaction proceeds in the cytosol of cells, where pregnenolone is transported from
mitochondria. The reaction is catalyzed by 3-(]-hydroxy steroid dehydrogenase. In
the membranes of endoplasmic reticulum 17-a-hydroxylase converts progesterone to
17-hydroxy-progesterone. The same enzyme catalyzes the conversion of pregnenolone
to 17-hydroxyprcgncnotone. A 17,20-lyases cleaves the two-carbon side chain
from 17-hydroxyprcgnenolone to form the -steroid, dehydroepiandrosterone.
9.3. Structure, Synthesis and Basic Action of Principal Hormones Regulating Fuel Metabolism 505

HO

Fig. 9.11. Synthesis of essential corticosteroids: 1 — conversion of cholesterol into pregnenolone:

2 — progesterone formation; 3 — progesterone nydroxyiatioti (17-21-11) and cortisol formation;
4 — progesterone hydroxylation (21-11) and me aldosterone formation; 5 — androgen syntnesis
pathway

17-hydroxy-progesterone is the precursor of cortisol and dehydroepiandrosterone is

the precursor of androgens. The enzyme 21-hydroxylase (P450-C2l)t localized in the
endoplasmic rcticulu, m membrane, catalyzes the hydroxyl al ion of 1.7-OH-progesterone
to 11-deoxycortisolc, which is transferred to the inner mitochondrial membrane and
hydroxylated by cytochrome P450-Cu to form cortisol. The rate of cortisol synthesis
and secretion is regulated by the hypothalamic-pituitary axis according to the negative
feedback mechanism (Fig. 9.13).
506 Chapter 9. Molecular enaocrmoiogy

Fig. 9.12. Steps of cortisol synthesis in ceiL The release of cholesterol from its esters and the synthesis
of corticosteroids are stimufatea by corticotropnin. The reactions of cortisol synthesis occur in
different cell compartments of me adrenal cortex

In blood, steroid hormones arc transported by specific transport proteins.

Catabolism of corticosteroids occurs primarily in the liver where reactions of
hydroxylation, oxidation, and reduction proceed. The products of the corticosteroid
catabolism arc conjugated with glucuronic acid and to smaller extent to sulfuric acid.
These conjugated compounds, termed 17-kctostcroids and 17-hydroxysteroids, arc
mainly excreted in urine.
Glucocorticoids arc the most potent regulators of fuel metabolism. Many of their
biological effects can be understood best as adaptations to sustained stressful situations.
9.3. Structure, Synthesis and Basic Action of Principal Hormones Regulating Fuel Metabolism 507

Cold exposure Exercise

Fig. 913. Regulation of cortisol secretion. Various factors act on hypotnaiamusto stimulate tne release
of CRH. CHR stimulates (+) tne release of ACTH from me anterior pituitary, vriiich stimulates (+) tne
release of cortisol from adrenal cortex. Cortisol inhibits (-) tne release of CRH and ACTH

Glucocorticoids interacting with specific cytosolic or nucleus receptors alter gene

transcription in target cells, change the amount of proteins, usually key metabolic
enzymes, that results in changing intracellular metabolic processes.
I n the liver, cortisol promotes glucose synthesis inducing the synthesis of the key
giuconcogcnelic enzyme, phosphoenolpyruvate carboxykinase (PEPCK) and the
enzymes of amino acid catabolism (alanine aminotransferase, tryptophan pyrrolase
and lyrosin aminotransferase). It increases protein breakdown in. skeletal muscle and
other extrahepatic tissues, which supplies amino acid precursors for gluconeogenesis.
In addition, cortisol stimulates glycogen synthesis in the liver and inhibits the
consumption of glucose by peripheral tissues.
Chronic cortisol excess promotes lipogenesis in. the certain regions of the body
(abdomen, trunk and face resulting in obesity). At the same time, it stimulates
lipolysis in the other regions (c.g., extremities) by increasing the lipolytic action of
catecholamines and growth hormone.
508 Chapter 9. Molecular eoaocrmoiogy

The glucocorticoid effect on the metabolism of proteins and nucleic acids is

manifested in two wavs. I n the liver, cortisol has mainly an anabolic effect by stimulating
the synthesis of proteins and nucleic acids. In muscles, lymphoid and adipose tissue,
skin and bones, cortisol has catabolic effect by inhibiting the synthesis of proteins,
RNA and DMA and stimulating the degradation of these macromolecules.
Al high concentrations, glucocorticoids suppress immune responses, causing the
death of lymphocytes and involution of lymphoid tissue: inhibit the inflammatory
reaction, reducing the number of circulating leukocytes, as well as inducing the
synthesis of lipoconins that inhibit phospholipase Ar thereby reducing the synthesis
of inflammatory mediators — prostaglandins and leukotrienes. Glucocorticoids
inhibit the growth, and division of fibroblasts, as well as the synthesis of collagen and
fibronectin. The typical signs of glucocorticoids hypersecretion are thinning of the
skin, slow wound healing, muscle weakness and muscle atrophy.
Glucocorticoids are involved in the physiological response to stress associated
with trauma, infection, or surgery. The catecholamines are primarily involved in this
response, but glucocorticoids in many cases stimulate them to exert maximum activity.
Mineralocorticoids stimulate reabsorption of Na' in the distal convoluted tubules
and collecting ducts of kidneys. In addition, they contribute to the secretion of
K', NH4 in the kidneys, as well as in other epithelial tissues such as sweat glands,
intestinal mucosa and salivary glands. In the human body, aldosterone is the most
active mineralocorticoid.
Epinephrine, norepinephrine and dopamine, or catecholamines, arc synthesized
from common precursor tyrosine in adrenal medulla that can be considered as an
extension of the sympathetic nervous system because preganglionic fibers from
the splanchnic nene terminate in the medulla to innervate chromaffin cells that
produce catecholamines. Such cells arc also found in the heart, liver, kidneys,
genital glands, postganglionic neurons of the sympathetic nervous system and in
the ccmral nervous system. Catecholamines arc stored in adrenal medulla in the
chromaffin granules. Their release from the adrenal medulla into bloodstream and
nerve endings is triggered by both nerve impulses and corticosteroids. Epinephrine is
most predominant catecholamine synthesized in the chroma Ilin cells of the adrenal
medulla. Epinephrine is transported to the liver and skeletal muscle. Norepinephrine
is mainly formed in organs innervated by sympathetic nerves (80% of the total} and
reaches peripheral tissues only in small amounts. The ha if-lifetime of catecholamines
is 10-30 s. The major portion of catecholamines is rapidly metabolized in various
tissues by specific enzymes. Only a small portion ofcpinephrinc (- 5%) is excreted in
the urine.
Catecholamines have multiple effects on gene expression and cellular [unction in.
the nervous, endocrine, cardiovascular, gastrointestinal, respiratory; reproductive, and
immune systems. They regulate cellular functions through distincL a-adrencrgic and
P-adrenergic plasma membrane receptors coupled to G-protein. All catecholamine
receptors are glycoproteins, which arc products of different genes, differ in affinity
for agonists and antagonists and transmit signals to cells using different secondary
messengers. This determines the nature of their effect on the metabolism in target cells.
9.4. Hormonal Regulation of Fuel MetaDolism at Normal Nutrition Rnytnm 509

Epinephrine interacts with both a- and B-receptors; in physiological concentrations,

norepinephrine interacts mainly with a receptors. The interaction of the hormone
with B (Bp Bp B->receptors activates adcnylyl cyclase, while binding to the a2 receptor
inhibits it. When the hormone interacts with the a-receptor, phospholipase C is
activated and the inositol phosphate signaling pathway is stimulated. The typical
feature of all biologicals effects of epinephrine and norepinephrine is the stimulation
of the processes in. the body to confront emergency situations, termed ^flight or fight*
response.

9A HORMONAL REGULATION OF FUEL METABOLISM AT NORMAL

NUTRITION RHYTHM
Mammalian survival demands an uninterrupted supply of metabolic fuels to
maintain body temperature, to escape from danger, and to grow and reproduce. A
constant supply of glucose and other energy-rich, metabolic fuels must be available to
the brain and other vital organs al all times despite wide fluctuations in food intake
and energy expenditure. Constant availability of metabolic fuel is achieved by storing
excess carbohydrate, fat and in special occasions protein principally in liver, adipose
tissue and muscle, and drawing on those reserves when needed. The most intense
How of substances in the body is associated with the use of carbohydrates and fats
(to a lesser extent amino acids) as energy sources. Glucose, fats of lipoproteins, laity
acids and ketone bodies arc the main energy carriers that arc distributed through the
bloodstream to the organs. Their main sources arc liver and fatty tissue; all organs use
these fuel molecules, and muscle tissue is the main consumer due to its considerable
mass and high energy intensity.
After a mixed meal, the digestion of carbohydrates is complete in about 2 hours,
the digestion of proteins and fats — in 4-5 hours (absorptive state), followed by a
postabsorptivc slate. In humans with three meals a day, it takes 10-15 hours per day
to digest. Energy consumption occurs during all 24 hours (with a certain decrease
during night sleep). Therefore, part of the energy during digestion is stored for use in
a postabsorptivc state.
The storage of nutrients is activated after eating and is replaced by mobilization
of reserves after the digestion. The transition from one state to another results in
signilicant metabolic changes. The deposition of carbohydrates (glycogen), the
deposition of fats, the predominant use of glucose to meet energy needs switches to
the mobilization of fuel reserves (glycogen and fats) and the predominant use of fats,
and also amino acids as energy sources.
in humans with three meals a day the changes of these stales occur three times a
day too. I?ut they arc not clearly expressed because the intervals between meals arc
rather short (5-6 hours). The post-absorptive state has hardly ti me enough to start,
when the next meal time comes. A typical postabsorptivc state is considered to be a
state before breakfast in. the morning after approximately ten hours after the previous
meal. One meal a day provides a clearer picture. During the day, glycogen stores in
the body arc exhausted, gluconeogenesis becomes the only source of glucose, glucose
is used primarily by nerve cells, while almost all other celts arc provided with energy
510 Chapter 9. Molecular endocrinology

due to oxidation of fatty acids, as well as ketone bodies formed in the liver from fatty
acids.
Liver, adipose tissue and muscles are the main organs that provide changes
in metabolism in accordance with the rhythm of nutrition. Many hormones affect
fuel metabolism including those that influence absorption, transport, and oxidation
of fuel molecules. Insulin is the major anabolic hormone: glucagon is the major
counterregulatory hormone. Epinephrine, cortisol, thyroid hormones, and growih-
hornione also have contrain.su la r activity. Insulin and contra instil ar hormones provide
a balance between the needs and capabilities of the body to obtain energy necessary for
normal functioning and growth.
Muscle activity slows down the storage processes during digestion, because a
part of the products of digestion coming from the intestine is directly consumed in
muscles. In the postabsorptivc stale, muscular activity stimulates the mobilization of
nutrient storage, mainly fats. Epinephrine plays an important role in the regulation
of metabolic changes in the body associated with the muscle work-to-rcst transition.
Metabolic changes of the major fuel molecules in the absorptive state arc mainly due
to the high insulin-glucagon index (Tig. 9.14).

cell

Fig. 9.14. Major metabolic pathways in absorptive state: 1 — glycogen biosynthesis in the liver; 2 —
glycolysis; 3 — TAG biosynthesis in me liver; 4 —TAG biosynthesis in adipose tissue; 5— muscle
glycogen biosynthesis; 6 — protein biosynthesis in various tissues, including the liver: FA — Fatty
acids
9.4. Hormonal Regulation of Fuel MetaDolism at Normal Nutrition Rnytnm 511

hi the liver, glucose consumption increases that is a consequence of the

metabolic pathways acceleration in which glucose is converted into deposited
forms of energy: glycogen, and fats. With an increase in glucose concentration in
hepatocytes, glucokinase is activated and converts glucose to glucose-6-phosphate.
In addition, insulin induces the synthesis of glucokinase mRNA. The hepatic
glucose-6-phosphate concentration increases that leads to a glycogen synthesis
speed-up in the liver. Simultaneous inactivation of glycogen, phosphorylase and
activation of glycogen synthase also contribute to this process. In hepatocytes,
insulin promotes glycolysis increasing the activity and the number of key glycolytic
enzymes: glucokinase, phosphofructokinase and pyruvate kinase. At the same
time insulin, inactivates the gluconeogenesis enzyme fructose-1,6-bisphosphatase
and represses ph os phoenol pyruvate carb oxykinase synthesis, thereby inhibits
gluconeogenesis. Increase in. the hepatic glucose 6-phosphate concentration in the
absorptive state and the active NAD PH use for the fatty acid synthesis stimulate
the pentose-phosphate pathway. The availability of substrates (acetyl-CoA and
NADPH) formed from glucose , and insulin-caused activation and induction of key
fatty acid synthesis enzymes accelerate fatly acid and TAG synthesis. Amino acids
entering the liver from the digestive tract arc used for synthesis of proteins and other
nitrogcn-containing compounds. The amino acid excess cither enters the blood
and is transported to other tissues, or is deaminated, followed by the oxidation of
nitrogen-free residues into the TCA cycle.
Adipose tissue mainly stores energy as triacylglyccrols. Insulin, facilitates the
glucose transport io adipocytes by activating GLUT-4. The increased intracellular
glucose concentration and activation of the key glycolysis enzymes provide the
formation of acetyl-CoA and glycerol-3-phosphate for TAG synthesis. Stimulation
of the pentose phosphate pathway ensures NADPH for the biosynthesis of fatty acids.
However, the fatty acids de now biosynthesis in human adipose tissue occurs at a high
rate only after a preceding fasting. With a normal nutrition rhythm, fatly acids used
for the TAG synthesis primarily are released from chylomicrons and VLDL under the
action of LP-lipase. The lipolysis is inhibited since the hormone-sen si Live TAG-lipase
is dephosphorylatcd and inactive in the absorptive state.
Insulin stimulates glucose transport into muscle cells because they are insulin
dependent as adipocytes and also contain GLUT-4. Glucose is phosphorylated and
oxidized to provide energy tor cells and is also used for glycogen synthesis. The amino
acid transport into muscles and protein biosynthesis is also accelerated by insulin,
especially after intake of protein-rich meals and during muscular activity.
In the postabsorptive state, the insulin/glucagon index decreases, and metabolic
changes arc mainly aimed to maintain the blood glucose concentration, which serves
as the major energy substrate for the brain and the only source of energy for red blood
cells. The basic metabolic changes occur in the liver and adipose tissue (Fig. 9.I5)
and provide replenishing glucose due to internal reserves and the use of other fuel
molecules (fatsand amino acids).
512 Chapter 9. Molecular endocrinology

Blood Liver

I Glucose
Cl .i I Insulin
f Glucagon

X Fattv .acidsAcetvI-CoA

5J
Ketone bodies

I ©
Ketone bodies

®I
TAG-----------

Adipose
cell

Urina
Muscles

Fig. 9.15. Major metabolic pathways in poslaDsorptive slate: I — decrease in We insuliri.'glucagan

ratio; 2 — glycogen breakdown; 3, 4 — glucose transport to we Drain and red mood cells; 5 —
lypotysls; 6 — TAG transport to the liver and muscles; 7—synfflesis of ketone Dodies in We liver: 8 —
transport of ketone Dodies to the muscles; 9 — gluconeogenesis from amino acids; 10 — synthesis
and excretion of urea; 11 — lactate transport to me liver for gfuconeogenesis: 12 — gluconeogenesis
from glycerol; KB — ketone body; FA — fatty acids

In the first few hours after a meal, the blood glucose level is diminished slightly,
and tissues receive glucose released from liver glycogen. Liver glycogen mobilization
is accelerated by glucagon. Glycogen stores in the liver arc depleted for the 24-
hour fast. As glycogen stores arc exhausted, the main source of glucose becomes
gluconeogenesis, which begins to accelerate 4-6 hours after the last meal. Substrates
for the glucose synthesis arc lactate, glycerol and amino acids. The rate of fatty
acid biosynthesis decreases due to phosphorylation and inactivation of acetyl-CoA
carboxylase, and the rate offJ-oxidation increases. In adipose tissue, the rate of TAG
synthesis decreases and lipolysis is stimulated. Stimulation of lipolysis is the result
of the activation of the hormone-sensitive TAG-lipase by glucagon. As lipolysis
increases during postabsorptivc state, fatty acids are released from adipose tissue and
travel to various organs. In parts these fatty acids arc taken by the liver for C-oxi dal ion.
9.4. Hormonal Regulation of Fuel MetaDolism at Normal Nutrition Rnytnm 513

Acetyl-Co A. produced in the liver, is convened to ketone bodies. Fatty acids become
important sources of energy in the Liver, musclesand adipose tissue. Thus, in the post­
absorption state, the blood glucose concentration is maintained at 60-100 mg/dL
(3.5-5.5 mmol/Lh and the level of fatty acidsand ketone bodies increases.

Review Tests
Match the figure and the letter.
1. Stages of the insulin biosynthesis and maturation. Com pern on ts:
A. N-terminal amino acid.
B. N-signal peptide.
C. Preproinsulin.
D. Proinsulin.
E. C-pep tide.

2. Biosynthesis of thyroid hormones. Proteins:

A. Thyroglobulin with DIT.
B. Thyroglobulin with T4.
C. ThyroperoKidasc.
D. Thyroglobulin.
E. Na4, K*-AT Rase.
514 Chapter 9. Molecular endocrinology

3. Scheme or regulation of the thyroid hormone synthesis and secretion. Hormones:

A. Ci HR.
B. ACTH.
C. lodothyronincs.
D. Thyroxine.
E. Thyrotropin I ibcrin.
Hypothalamus

Pituitary

Thyroid
9.4. Hormonal Regulation of Fuel MetaDolism at Normal Nutrition Rnytnm 515

4. Synthesis of basic corticosteroids. Steroid:

A. Testosterone.
B. Aldosterone.
C. Pregnenolone.
D. Progesterone.
E. Cortisol.

Situational Problems
I. A student, halfan hour after the dinner, containing about 150 g of carbohydrates,
20 g oflat, and 40 g of protein, is silling in a chair and reading. What metabolic
changes occur in this state? For answer:
a) name the hormone level in blood that is elevated during resting:
describe the steps of its synthesis, name the secretion stimuli and target tissues;
b) present a diagram of the hormonal signal transduction to target cells:
c) draw diagrams reflecting the student's metabolic changes of glucose and fats
in adipocytes:
d) enumerate the regulatory enzymes of fatty acid synthesis from glucose that
have their activity and amount increased under the in fluence ofthe mentioned
hormone.
516 Chapter 9. Molecular eoaocrmoiogy

2. in die sports camp, a group of students have been intensively swimming in

a pool for 60 minutes lwo hours after each meal. What metabolic changes of
carbohydrates can be assumed in students during physical exertion from its start
to completion? To answer:
a) name the hormone with its blood concentration increased during physical
activity, name the site of its synthesis, the stimuli of secretion, the target
tissue, and draw a diagram of the hormonal signal transduction to target cells;
b) describe the changes in carbohydrate metabolism in the liver and muscles of
students during workouts, and present appropriate metabolic pathways:
c> enumerate the fuel molecules that provide muscles with energy at different
moments of students’ physical exertion.
3. In the sports camp, a group of students have been intensively swimming in a
pool for 120 minutes two hours after each meal. What metabolic changes of the
main energy carriers can occur in students after prolonged physical exertion?
For answer:
a) name the hormones with their secretion to blood increased during prolonged
muscle activity: indicate the place of their synthesis, chemical nature,
secretion stimuli, target organs, main biological effects.
b) present a diagram describing the action mechanism of one of the hormones
on target cells beta receptors;
c) describe the features of signal transduction for another hormone that interacts
with receptors associated with cytoplasmic protein kinases:
d) list the metabolic changes of carbohydrates, fatsand proteins in the liver and
muscles caused by these hormones during exercise.
4. During a lunch at a McDonald’s outlet, an office employee received about 350 g
of carbohydrates with food. How will his metabolism of carbohydrates and fats
in the liver change two hours after meal? For answer:
a? name the hormone with increased level during the absorptive state, enumerate
the main stages of its synthesis, their localization, target organs, the structure
of the receptor and the mechanism of hormonal signal transduction to the
target cells:
a) list the ways of glucose catabolism, that arc activated in the liver during this
state, enumerate the ways the glucose catabolism products arc used;
b) write the formulas for the regulatory reaction of fatty acid synthesis, name the
key enzyme and describe its activation mechanisms:
c) draw a chart for fatty acids synthesis;
d) describe the subsequent ways of using fatly acids synthesized in hepatocytes.
5. A young woman, decided to lose weight and abstained from fat-containing food
for several months. Her diet contained an increased amount of carbohydrates
relative to the norm, and her weight increased. Explain the reason for weight
gain in this case. For answer:
a) name the daily intake of carbohydrates with normal nutrition and explain
how the insulin/glucagon ratio changes in this situation;
b) draw a diagram of fat metabolic pathway with its elevated rate that led to
increased weight;
9.4. Hormonal Regulation of Fuel MetaDolism at Normal Nutrition Rnytnm 517

c) draw a diagram of the fatty acid metabolic pathway that has its rate increased
under these conditions;
d) describe the process regulation mentioned in c), write the regulatory reaction
of this process, name the key enzyme, its activatorsand inhibitors;
e) name all metabolic pathways of glucose that provide the precursors formation
for the fatty acids synthesis, and list all needed components formed from
glucose.
6. With the presence of fast-food chains, many people have the opportunity to
«havc a quick bite* while their daily physical activity is decreased. Explain why
such a diet leads to obesity more quickly under hypodynamic conditions. For
answer:
a) draw the chans of metabolic pathways in adipocytes with their activation
leading to increased weight:
b) give the origin of substrates, enzymes, ways of using final products:
cidraw an appropriate chart and describe the stages of sequential hormonal
signal transduction to adipocytes and explain the biological effects of the
hormone.
7. After visiting a gym, a student had 250-300 g of carbohydrates for lunch and
decided to rest. How will the student’s metabolism of carbohydrates and fats in
the adipose tissue change 2 hours after meal?
a) draw a graph displaying the student’s blood glucose concentration over time
during the absorptive state; explain the curve;
b) name the hormone that has its level elevated in the student’s blood in the
absorptive state and describe the hormonal signal transduction to adipose
tissue;
c) list all metabolic pathways of glucose utilization and catabolism products in
adipose tissue and explain their physiological significance;
d) draw’ a chart of the fat metabolic pathway in adipoesies and explain how the
hormone stimulates the process.
S. A patient suffering from infectious polyarthritis, has been receiving prednisone
for a long time as treatment. Prednisone is a structural analogue of cortisol. As
the patient fell better, he deliberately stopped taking this d rug. Soon the patient’s
state look a dramatical turn for the worse. Screening showed a decrease in the
patient's blood glucose concentration, the blood pressure dropped, and the
content of 17-kctostcroids in the urine was diminished. Why did the patient’s
state worsen after he quitted this drug? For the answer:
a) draw a flowchart of hormone synthesis and secretion for the hormone with
inhibited production caused by the prolonged administration of prednisone:
b) predict an improvement in the patient’s state if he is given corticotropin;
c) name the reasons for the blood glucose concentration, and 17-kctostcroids
decrease: and blood pressure decline; write the metabolic pathway with its
decreased rate in the liver when prednisone administration was stopped;
d) substantiate the prednisone use to treat arthritis.
9. A 46-year-old woman visited a doctor with complaints of sweating, rapid
heartbeat, general weakness and fatigue. The patient also told the doctor that
3-4 hours after eating, she has been experiencing acute bouts of hunger, with
clouded consciousness, speech disorders, spatial and temporal disorientation.
518 Chapter 9. Molecular eoaocrmoiogy

Moreover her body weight. has been constantly increased for the past few
months. Screening showed the blood glucose level was 2.8 mol/L, the insulin/
glucagon ratio is 0.7 (norm >0.4), the C-peptide level was elevated. What allows
to suspect insulinoma (endogenous hyperinsulinism) in this woman? Please,
answer the following questions:
a) what hormone is high secreted in this condition?
b} what is the possible cause of this disease?
c) why do people with such condition gain weight? Draw a diagram of the
metabolic pathway reflecting the cause of excess weight.
d) what is the role of this hormone in the regulation of this process?
e) what treatment is needed lor this disease?
10. Symptoms of insulinoma, a hormone-producing tumor of the 0-cclls of the
islets of Langerhans of the gastric gland, manifest themselves in increased
blood insulin levels, bouts of hypoglycemia, accompanied by general weakness,
fatigue, sweating, tachycardia, loss of consciousness, and acute hunger. Explain
the causes of the disease symptoms. For that:
a) name the normal blood glucose concentration:
b) draw a chart of an insulin receptor and describe the steps of insulin signal
transduction to the target cells;
c) recount the features of glucose transport into cells of different tissues, name
the insulin-dependent tissues;
d) explain bouts of hypoglycemia in insulinoma, and draw the metabolic
pathways of glucose metabolism with increased speed in the liver that arc
associated with this condition.
11. A 45-year-old man complained of rapid weight loss, tachycardia, increased
sweating, occasional elevated blood pressure and increased excitability.
Additional screening revealed a tumor in the medulla of one of the adrenal
glands. The patient was diagnosed with pheochromocytoma. What molecular
mechanisms caused the development of the condition showing the sc symptoms?
For answer:
a) draw a chan of the catecholamines synthesis in adrenal glands;
b) name the hormone that has its synthesis increased in. this disease, list its
target organs, stimuli of synthesis and secretion, target tissues and receptors;
c) draw a chart of signal transduction of this hormone to adipocytes and the
metabolic pathway of fat with i ncreased rate in. the ^patient’s adipocytes;
c) list the physiological effects of this hormone and explain the causes of the
disease symptoms.

93. METABOLIC CHANGES DURING STARVATION

A healthy adult human reserves the fuel as glycogen in the liver and, in relatively
small amounts, in muscles; large quantities of fats in adipose tissues: and tissue
proteins, which can be degraded wrhcn necessary eo provide fuel.
More than 24 hours a Iler the last meal blood glucose falls further to the lower
limit of normal, insulin secretion is slowed, secretion of glucagon and GH is increased
9.5. MetaDoiic Changes During Starvation 519

and the insuJin/glucagon ratio is reduced. Cortisol secretion follows its basal circadian
rhythm and it's level remains almost unchanged until prolonged hypoglycemia results
in increased blood cortisol concentration in the late stages of fasti ng. These hormonal
signals designate the predominance of triacylglyccrols mobilization and protein
degradation and exhaustion of glycogen, stores. Fatty acids now become the primary
fuel for muscle and liver.
In this state, contra insular hormones regulate the flux ofsubstrates be tween the liver,
adipose tissue, musclesand the brain to maintain the blood glucose: concentration due
to gluconeogenesis to ensure glucose-dependent tissues brain, red blood cells): and to
mobilize other fuel molecules, primarily fats, to provide energy to all other tissues. The
manifestation of these changes allows us to conditionally distinguish three phases of
starvation. Due to the switch of metabolism to fuel mobilization, even after 5-6 weeks
of fasting, the blood glucose glucose levels arc still in the range of 65 mg/dl. The main
changes during lasting occur in the liver, adipose tissue and muscles {Fig. 9.I.6).

Blood Liver

tGhjeose Glycogen
I Insulin
ffGhJcagon
[Cortisod Glucose

Glycerol

Brain

Lactale

Ketone bodies

.Amino acids

Adipose
cell

Kidney

Muscles

Fig. 9J6. Metabolic Changes of me main energy sources during fasting: 1 — decrease in me insulin/
glucagon ratio; 2 — glycogen mobilization; 3,4 — glucose transport to the Drain and red blood cells;
5 — TAG mobilization; 6 — FA transport io the muscles; 7 — synthesis of ketone bodies; 8 — FA
transport to the liver; 9—Amino acid transport to the liver; 10 — gluconeogenesis from amino acid;
11 — lactate transport to the liver; 12—glycerol transport to me liver. Dotted lines indicate processes
mat are slowing
520 Chapter 9. Molecular enaocrinoiogy

Phases of fasting. Fasting may be short-term — during the day (first phase) T
continue for a week (second phase) or several weeks (third phase). During fasting, a
number of changes in fuel metabolism occur.
During the first phase, the blood glucose concentration decreases to approximately
60 mg/dl, leading to a decrease in the blood insulin level by about 10-15 times
compared with the absorptive state, and a rise in the blood glucagon, level.
The changes in hormonal status and the action of intracellular regulatory
mechanisms result in glycogen stores in the body to be almost completely depleted
by a 24-hour fasting, and the rate of fat mobilization and the rate of gluconeogenesis
increase. The liver glycogen is converted back to free glucose to maintain normal
blood glucose levels between meals, thus, only short-term fasting is provided by
the liver glycogen mobilization. The only process that provides tissue with glucose
during long-term fasting is gluconeogenesis. Gluconeogenesis begins eo accelerate
4-6 hours after the last meal and becomes the only source of glucose during
prolonged fasting period, maintaining the blood glucose at lower limit of the normal
in subsequent periods of fasting. Amino acids, glycerol and lactate arc the main
substrates of gluconeogenesis.
During the second phase, a decrease in the blood glucose level keeps low insulin/
glucagon ratio and leads to increased secretion of other counted nsular hormones,
cortisol and growth hormone. During the first days of lasting tissues consume
less glucose than they use in postabsorptive slate. The only source of glucose is
gluconeogenesis from amino acids and glycerol. Although body proteins arc not
primarily a reserve form of fuel as glycogen or fats, during the first few days of fasting
muscle proteins arc quickly degraded into amino acids which become the main
carbon source for glucose synthesis. The energy needs of the muscles and most
other organs arc met principally by fatty acids and ketone bodies, because the blood
insulin concentration is very low and glucose does not enter muscle cells. The rate
of TAG mobilization continues to rise, the blood fatty acid concentration increases
approximately twice compared with the postabsorptive stale. The rate of ketone bodies
formation in the liver increases significantly driven by low instilin/ghicagon ratio and,
accordingly, their blood concentration increases too (Fig. 9.17).
After 3-5 days of fasting the concentration of ketone bodies in the blood reach 2
to 3 mMol, and exhaled air and sweat of the fasting person smells of acetone. Under
these conditions, only insulin-in dependent cells, and especially brain cells, become
glucose consumers. However, ketone bodies begin to provide for a notable portion of
the metabolic needs for brain and other nervous tissues and, consequently, these tissues
oxidize less glucose too. The only source of glucose is gluconeogenesis that continues
due to amino acids derived from the breakdown of tissue proteins and glycerol from
adipose triacylglyccrols. After a week of fasting metabolic rale is generally reduced and
oxygen consumption decreases by about 40%.
During the third phase, the metabolic rate continues to stow down. The rate of
protein breakdown decreases, and after several weeks of fasting, it stabilizes at about
20 g per day. When this amount of proteins is broken down, about 5 g of urea per day
is formed and secreted (at normal nutrition rhythm — 20-25 g). The nitrogen balance
9.6. Metabolic Changes During Muscle Activity 521

Fig. 9.1T Changes in blood fuels during fasting. The plasma levels of fatty acids and ketone Dodies
increase in starvation, whereas mat of glucose decreases

in all phases of fasting is negative. The rate of gluconeogenesis from amino acids is
reduced. In this phase, the brain continues to use both ketone bodies and glucose, bin
less actively due to a decrease in the rate ofgluconeogenesis and a decrease i n the blood
glucose concentration. A decrease in the level of gluconeogenesis from amino acids is
necessary to preserve protein, since severe protein loss (loss of 1/3 of all proteins,
including heart muscle mass) causes a malfunction of the major organs and can lead
to death. Continued slow protein loss during fasting of an extremely obese person can
lead lo death, from protein depletion even before TAG stores are depleted. The rate of
oxidation of ketone bodies in the muscles is reduced, and they almost exclusively use
fatty acids. Almost all of the body's energy needs are met by oxidizing fatty acidsand
ketone bodies until TAGs are depleted. The duration of starvation depends on how
long ketone bodies can be synthesized and oxidized to produce ATP. The oxidation of
the ketone body requires oxaloacctatc and other components of the TCA cycle, which
are formed from glucose and amino acids at a normal rhythm of nutrition, and when
starvation only from amino acids.

9.6. METABOLIC CHANGES DURING MUSCLE ACTIVITY

Both, at rest and during muscular activity, glucose is the main fuel for the central
nervous system of mammals. Any physical activity is accompanied by an increase
in muscle metabolism and subsequent partial use of blood glucose. Fuel needs arc
met by mobilizing reserves in muscle cells, as well as by extra-muscular fuel reserves.
Rapid absorption of glucose from the blood can dangerously reduce the blood
glucose concentration. Normal blood glucose levels during muscle activity require
strict glucose regulatory mechanisms and can be maintained by increasing glucose
production and release by stimulating the breakdown of liver glycogen and stimulating
522 Chapter 9. Molecular eoaocrmoiogy

the glucose synthesis from lactate, amino acidsand glycerol, as well as by mobilizing
other types of fuel, which can serve as an alternative (fatty acids, ketone bodies). The
endocrine system in these conditions is important primarily for the maintenance or
replenish me nt of fuel in the muscles.
Du ring short-term muscular activity, the main sources of energy that ensure muscle
functioning arc endogenous .ATP stores in muscles, creatine phosphate and glucose
from glycogen. The total ATP content in the muscle is approximately 5 pmol per I g
of its mass that is enough only for about I. s muscular activity. A high-energy molecule
creatine phosphate is reversibly formed from creatine and ATP under the action of
creatine kinase (Ch. 8). The level of creatine phosphate in resting muscle is several­
fold higher than that of ATP. This stores provides intensive muscle work for 2-5 s
due to rapid ATP rcsynthesis by substrate-level phosphorylation using the high-energy
creatine phosphate and ensures a person to run 15-50 meters. As creatine phosphate
stores are depleted, glucose from muscle glycogen becomes a major source of energy.
For short-term maximum efforts, energy is released from the fuel anaerobically, before
regulation of the blood circulation can provide the necessary oxygen. Epinephrine and
norepinephrine, released from the adrenal medulla and sympathetic nerve endings in
response to central activation of the sy mpathetic nervous system, stimulate glycogen
degradation. The breakdown of glycogen lo glucose and the glucose oxidation to
lactate provide the required ATP. Calcium released from the sarcoplasmic reticulum
in response to nerve stimulation not only causes muscle contraction, but also activates
glycogen phosphorylase. Lactate released from working muscles is converted to glucose
in. the liver and can be exported back to the muscles in the Cory cycle. More long-
duration muscle activity requires adequate oxygen delivery and the capacity for the
muscle to utilize the oxygen delivered. During exercises overall oxygen consumption
may increase 10- to 15-fold in a well-trained athlete. The relative intensity of the
anaerobic and aerobic glycolysis in this ease changes. The rate of anaerobic glycolysis
decreases while the rate of aerobic glycolysis increases.
Glucose is also an important fuel in the early stages of moderately intense exercise.
As physical exercises continue, skeletal muscles significantly deplete their own glycogen
stores and begin to compete with the brain for the glucose available in the blood (for
example, glucose released from the liver}. Hut during prolonged physical exercises, the
muscles begin actively to use fatly acids as fuel molecules. As the duration of physical
exercise increases and the level of insulin decreases in combination with an increase in
the contra-insular hormones level, the lipolysis enhances that releases fatty acids from
adipose tissue. Maintaining low insulin levels allows laity acids lo circulate in higher
concentrations available for tissue use, sparing glucose. Fatty acids arc transported
lo the working muscles and liver. In muscles, they arc actively used for the oxidation
and synthesis of ATP. In the liver, fatty acids are oxidized to acetyl-CoA, which, arc
the precursors for the ketone body synthesis during prolonged physical load. Ketone
bodies provide useful fuel for various tissues, including the brain, while reducing the
blood glucose level.
The combined effects of the fall in insulin secretion and the rise in glucagon
secretion arc complemented by the contribution of catechol a mines, epinephrine and
norepinephrine, and in the liver they increase glycogenolysis and gluconeogenesis,
9.7. Metabolic Changes in Hypo- ana Hypersecretion of Hormones 523

while simultaneously stimulating muscle glycogenolysis and lipolysis in adipocytes.

Initially, the contributions of in creased secretion ofGH and cortisol to the production
of hepatic glucose arc insignificant, but during prolonged exorcise, the contributions
or both increase due to the induction of gluconeogenic enzymes in the liver.
During prolonged workouts a rather important source of glucose for working
muscles can also be glycogen stores in non-working muscles. Epinephrine and
norepinephrine stimulate glycogenolysis in both, working and non-working muscles.
Glucose-6-phosphate that is formed during glycogen degradation can be completely
split into carbon dioxide and water in working muscles. Non-working muscles turn
it into pyruvate and lactate, which enter the blood and are taken by the liver to be
converted into glucose. Glucose returns to the blood circulation and selectively
captured by working muscles.

9.7. METABOLIC CHANGES IN HYPO- AND HYPERSECRETION

OF HORMONES
A change in the rate of hormones synthesis a nd secretion can occur as an adaptation
to a change in the physiological activity of the body, but is often the result of impaired
functional activity of the endocrine glands during the development of pathological
processes or abnormalities of regulation. Those disorders can occur cither in. the form of
hypofunction, leading Loa decrease in the amount of the hormone, or hypcrfuncLion,
accompanied by its excess synthesis.
CH deficiency occurs in. children and adults. In children, this condition is known
as hypopituitary dwarfism and manifested by growth failure that results from lack
of GH during childhood. GH deficiency in children is fasting hypoglycemia, likely
attributable to impaired substrate mobilization for gluconeogenesis and enhanced
insulin sensitivity. Pituitary dwarfs typically are of normal weight and length at birth
and grow rapidly and nearly normally during early infancy. Before the end of the first
year growth is noticeably below normal and conti nues slowly for many years. Typically,
the pituitary dwarf retains a juvenile appearance because of the retention of«baby fat*
and the disproportionately small size of maxillary and ma nd ibular bones. Symptoms of
chronic adult GH deficiency syndrome include increased body fat, decreased muscle
bulk and lean body mass, osteoporosis, reduced sweating, dry skin, and psychological
problems.
Hypersecretion of growth hormone is a condition termed as acromegaly in adults
and a giantism in children. In adult, acromegaly usually occurs in the 40-59-ycar-old
age group and results from continuous exposure Lo high levels of GH and I GF-1 and is
al most always caused byaGH-secreting pituitary adenoma. I n children and adolescents
whose epiphyseal plates have not yet closed, the effect of increased GH levels causes
excessive skeletal growth. Overproduction of GH in she adult, when epiphyseal closure
has occurred and increased amounts of GH and IGT-I. causes connective tissue
proliferation and increased cytoplasmic matrix, as well as bony proliferation, results
in the characteristic appearance of acromegaly. There is thickening of the cranium,
the mandible, and enlargement of some facial bones and bones in the handsand feet,
524 Chapter 9. Molecular eoaocrmoiogy

and elongation of the ribs gives a typical barrel-chested appearance. In acromegalic

patients there is also thickening of the skin and dis- proportionate growth of some soft
tissues including spleen and liver. The increased GH levels also has significant effects
on glucose, lipid, and protein metabolism. Hyperglycemia results from inhibition
of peripheral glucose uptake, and increased hepatic glucose production. Diabetes
mcllitus may occur when the pancreas cannot secrete enough insulin to offset the
effects ofGH. Excessive levels of GH and IGF-1 also affect the cardiovascular system.
Hyperfunction of the thyroid gland (hyperthyroidism) is manifested in several
clinical forms. Diffuse toxic goiter (Graves’ disease. Graves disease) is the most
common thyroid disease. In this disease, there is abnormal enlargement of the thyroid
gland (goiter),an increase in. the thyroid hormone concentration by 2-5 timesand the
development of thyrotoxicosis.
Typical signs of thyrotoxicosis can include an increase in basal metabolic rate,
increased heart rale, muscle weakness, loss of body weight (despite increased
appetite), sweating, fever, tremor and exophthalmos (eye patch). These symptoms
re fleet the simultaneous thyroid hormone stimulation of both anabolic (growth and
differentiation of tissues) and catabolic processes (carbohydrate, lipid and protein
catabolism). The catabolic pathways arc intensified to a greater extent, as evidenced by
the negative nitrogen balance. Hyperthyroidism can occur as a result of a tumor, an
inflammation (thyroiditis), and excessive iodine and iodine-containi ng drugs intake,
as well as autoimmune reactions.
Autoimmune hyperthyroidism is caused by the antibodies formation to thyroid-
stimulating hormone receptors in the thyroid gland. One of them is immunoglobulin
(IgG) mimics the thyrotropin action, interacting with the TSH receptor on the thyroid
cell membrane. This leads to diffuse proliferation of the thyroid gland and excessive
uncontrolled T,and T4 production, since the IgG formation is not feedback regulated.
The TSH level in this disease is reduced due to the suppression of pituitary function
by high thyroid hormone concentrations.
Hypothyroidism can be caused by an insufficient iodine intake (endemic goiter).
Less commonly, hypothyroidism occurs as a result of congenital defects of enzymes of
the thyroid hormone synthesis (c.g., thyropcroxidasc), or as a complication of other
diseases with damaged the hypothalamus, pituitary, or thyroid gland. In some forms of
hypothyroidism, antibodies to thyroglobulin arc detected in the blood. Hypofunction
of the thyroid gland in early childhood leads to cretinism, a delay in physical and
mental development. In adults, hypofunction manifests as myxedema (mucosal
edema) because of both the impaired metabolism of intercellular matrix molecules,
which are polyanions and have increased hydrophilicity, and the increased capillary
permeability. The main manifestation of myxedema is the excessive accumulation of
proteoglycans and water in the skin. The main symptoms of hypothyroidism include
drowsiness, decreased tolerance to cold, weight gain, decrease in body temperature.
Hypercortisol ism means a condition with chronically high blood corticosteroid
levels, mainly cortisol, and is often resulted from a violation of the regulatory
mechanisms of cortisol synthesis:
► with pituitary tumors and increased corticotropin production (I tsenko-Cushing’s
disease):
9.7. Metabolic Changes in Hypo- ana Hypersecretion of Hormones 525

► with adrenal tumors and increased cortisol production (Itscnko-Cushing’s

syndrome).
One of this condition symptoms is glucose tolerance decrease, that is, excessive
hyperglycemia after a meal or glucose load. In severe cases, hyperglycemia also
occurs in a post-absorptive state. The blood glucose concent radon may exceed the
renal barrier, and then glycosuria occurs (glucocorticoid-induced diabetes melliius).
Reduced glucose tolerance and hyperglycemia arc associated with increased protein
catabolism and gluconeogenesis from amino acids. Polyuria may be a manifestation of
hyperglycemia and resultant glycosuria.
The catabolic effects of cortisol on peripheral tissues causes protein degradation that
leads to muscle wasting and muscle weakness and is especially obvious in the musclesof
the extremities, with thinning of the limbs. Cortisol inhibits the activity and synthesis
of certain enzymes involved in the collagen and glycosaminoglycans formation. Loss
of collagen leads to thin, weakened integumentary tissues through which capillaries arc
more visible and makes small vessels susceptible lo rupture, loading to easy bruising,
even with minor trauma. For this reason, the atrophy of the skin occurs at the sites
of prolonged cortisol administration. The collagen synthesis in the bones is also
disrupted, and as a result, the inclusion of calcium and phosphate salts in bone tissue
is disturbed, and in this case a negative balance of these salts occurs. Osteoporosis,
a serious consequence of endogenous hypcrcortisolism. with a change in bone tissue
composition, mainly mineral, and increases in bone resorption and a loss of the protein
matrix, can results in pathologic fractures, vertebral compression fractures, bone and
back pain, kyphosis, and reduced height. In very high levels of ACTH, bronze or
brownish hyperpig mentation of the skin, mucous membranes and hair occurs. This is
caused by increased levels of melanocyte-stimulating hormones resulting from excess
proopiomelanocortin synthesis when ACTH concentration is elevated.
With elevated cortisol levels, vascular sensitivity to catecholamines is increased
significantly, leading to vasoconstriction and hypertension. Hypertension almost
always accompanied hypcrcortisolism and is partly caused by increased production of
another hormone of the adrenal cortex — aldosterone (hyperaldosteronism). It should
be noted that all corticosteroids have a mixed effect. In particular, cortisol not only
stimulates gluconeogenesis, but also causes sodium retention. Cortisol is hundred
times less active than aldosterone, however, in higher blood concentration (by two
orders of magnitude), cortisol can be thought to make a significant contribution to the
development of hypertension.
Hypoconisolism (low levels of cortisol secretion) can be primary, secondary’, or
tertiary. Primary hypocortisol ism (Add ison/s disease) develops because of an inability
of the adrenals to produce and secrete the adrenocortical hormones as a result of
damage to the adrenal cortex by a tuberculosis or autoimmune process. The main
clinical manifestations arc weight loss, general weakness, decreased appetite, nausea,
vomiting, decreased blood pressure and skin hyperpig men tat ion typical of primary
adrenal insufficiency Gbronze disease*). The cause of hyperpig mentation is increased
production of POM K, the precursor of ACTH and melanocyte-stimulating hormone.
Secondary adrenal insufficiency can. develop with ACTH deficiency. In secondary
adrenal insufficiency, unlike Addison's disease, there is no hyperpig mentation.
526 Chapter 9. Molecular eoaocrmoiogy

Tertiary hypocortisolism is commonly caused by abrupt withdrawal of exogenous

glucocorticoids or as a complication of treatment for Cushing’s syndrome. The long­
term corticosteroid therapy decreases hypothalamic corticotropin-re leasing hormone
(CRH) synthesis and secretion by the feedback mechanism, and also block the trophic
and ACTH-secretogogue actions of CR H on anterior pituitary cells, resulting in loss
of ACT H and its actions on the adrenal gland.
The absence of stimulating signals leads to atrophy of the adrenal cortex cells.
With the abrupt hormonal drugs withdrawal, acute adrenal insufficiency (the so-
called ^withdrawal syndrome*) may develop, which is a greater threat to life, as it is
accompanied by decompensation of all types of metabolism and adaptation processes.
It is manifested by vascular collapse, severe weakness, loss of consciousness. This
condition occurs owing to electrolyte metabolism, which leads to the loss of Na'
and Cl ions with urine and dehydration following the loss of extracellular lluid.
The change in carbohydrate metabolism is manifested in a decrease in blood glucose
levels, a decrease in glycogen stores in the liver and skeletal muscles. Symptoms and
management arc similar to those for secondary hypocortisolism.
Hereditary adrenogenital dystrophy in 95% of eases is the result ofa 21 -hydroxylase
deficiency (Fig. 9.9). This increases 1.7-0H progesterone and androgen production.
Characteristic symptoms of the disease involve early puberty in boys and the
development of male sexual characteristics in girts. A partial failure of 21-hydroxylase
can result in menstrual irregularity in women.
Hyperfunction of the adrenal medulla is caused by pheochromocytomas or
sympathetic paragangliomas that produce and release excessive amounts of
catecholamines. Majority of these tumors arc located in the adrenal medulla and
produce both epinephrine and norepinephrine. Tumors in extra-adrenal locations
produce only norepinephrine. Chronic c (Teets of catecholamine secretion include
persistent hypertension, headache, sweating palpitations, tachycardia, palpitations,
and pallor. Glucose intolerance may occur because of catecho la mine-induced
inhibition of insulin release by the pancreas.

9.8. METABOLIC CHANGES IN DIABETES MELLITUS

Diabetes mcllitus is the most common, serious metabolic disease in the world
occurring as a result of absolute or partial insulin deficiency. According to the WHO
classification, two main forms of the disease arc distinguished: type I diabetes —
insulin-dependent diabetes mcllitus (IDDM), and type II diabetes — non-insulin-
dependent diabetes mcllitus (NIDDM).
IDDM is a consequence of the destruction of the insulin-secreting P-eel Is of
the islets of Langerhans in the pancreas as a result of autoimmune reactions. A viral
infection such as viruses of smallpox, rubella, measles, cytomegalovirus, mumps,
Coxsackie virus, adenovirus causes the destruction of p-cells and can. provoke the
onset of type 1 diabetes. IDDM accounts for approximately 10% of the diabetic eases
worldwide.
As a rule, the destruction of p-cells occurs slowly and the onset of the disease
is not accompanied by metabolic derangements. When 80-95% of the cells die, an
9.8. MetaDoi ic Changes in Diabetes Melliius 527

absolute insulin deficiency occurs and severe metabolic disorders develop. In most
eases 1DDM usually begins before age 20, affecting children, adolescents and young
people, bin can occur al any (starting from I-year-old) age.
NIDDM develops as a result of various causes involving impaired conversion of
proinsulin to insulin, regulation of insulin secretion, increased insulin catabolism,
damage to insulin signal transmission mechanisms in target cells (for example, insulin
receptor defect, damage to intracellular insulin signal mediators, etc.), the formation
of anti bodies to insulin receptors (and the concentration of insulin in the blood may be
normal or even elevated). The factors determining the development and clinical course
of the disease include obesity, improper diet, sedentary lifestyle, stress. NIDDM
typically arises later in life than docs the insulin-dependent form a fleeting people,
usually over40 years old. develops gradually, the symptoms arc moderately expressed.
Acute complications arc rare.
in diabetes, as a rule, the insulin/glucagon raLio is reduced. At the same time, the
stimulation of glycogen and TAG synthesis is reduced, and mobilization of the energy
reserves is intensified. Even after a meal, the liver, muscles, and adipose tissue function
in a postabsorptivc mode. Moreover, products of digestion, as well as their meta bo I i Les,
instead of being deposited in the form of glycogen and fat. circulate in the blood.
All forms of diabetes arc characterized by an increase in the blood glucose
concentration — hyperglycemia, both after meals and on an empty stomach, as well as
glycosuria. In. normal individual a fasting blood glucose concentration is maintained
al around 90 mg/dL of plasma (5mM). After a meal, the blood glucose levels return
to baseline values within two hours, and the peak value docs not rise above 140 mg/
dL(7.8 mMol/L). In diabetes blood glucose levels rise much higher and maybe 300-
500 mg/dl and remains high at the post-adsorptive state (reduced glucose tolerance).
A decrease in glucose tolerance is also observed in cases of latent diabetes mcllitus.
In these cases, people have no complaints and clinical symptoms characteristic of
diabetes, and the fasting blood glucose concentration corresponds to the upper
limit of normal. However, the use of provocative tests (for example, sugar load)
reveals a decrease in glucose tolerance (Fig. 9.18). A fasting blood sample is taken
to establish a baseline glucose level. Then a patient drinks a glass of concentrated
glucose solution (75 g of glucose arc dissolved in 250 to 300 ml of water). To confirm
suspected diabetes, blood is drawn again after two hours and the blood glucose level
is measured.
The increase in blood glucose concentration, in IDDM is caused by several
factors. With a decrease in the insulin-glucagon index, the effects of contra insular
hormones increase, the number of glucose transporters (G LU T-4) on the membranes
of insulin-dependent cells (adipose: tissue and muscles) decreases. Consequently,
glucose consumption by these cells is reduced. In muscles and liver glycogen synthase
is phosphorylated and inactive; and glucose is not deposited as glycogen. In liver and
adipose tissue, the glycolytic enzymes and pyruvate dehydrogenase arc inactive and,
consequently, the conversion ofglucose into acetyl-CoA required for the TAG synthesis
declines, thus the rale of lipogencsis decreases. In addition, contrain.sular hormones,
primarily glucagon, activates gluconeogenesis from amino acids, glycerol and lac talc.
Thus increased hepatic production of glucose is combined with diminished peripheral
528 Chapter 9. Molecular enaocrmoiogy

utilization. The kidney glomerulotubular unit is capable of keeping glucose from

entering the urine until the scrum glucose level exceeds 175—185 mg/dl or 10 mM/L
(the tubular threshold for glucose). If blood glucose levels exceed this threshold,
glucose appears in the urine — glycosuria.

Fig. 9.13.Glucose intolerance test Bload glucose curves of a normal and a diabetic individuai after oral
administration of glucose solution. A criterion of normality is me return of tne curve to me initial value
witnin 2 nours. 1 — in a rteaitny person; 2 — in a patient wim diabetes

Ketoneniia is a characteristic symptom of diabetes. With a low insulin/glucagon

ratio, fats arc not deposited, their catabolism accelerates, since hormone-sensitive
lipase in adipose tissue is in a phosphorylated active form. The concentration ofnon-
estcrified fatty acids in the blood increases. The liver captures latty acids and oxidizes
them to acetyl-CoA, which in turn arc converted toacctoacctatc, 3-p-hydroxybutyrate,
and acetone, resulting in elevation of the blood ketone body levels — kctoncmia. In
the tissues, accloacctalc is partially decarboxylated to acetone, the smell of which
comes from patients with diabetes mellittis and is felt even from a distance. Increasing
the concentration of ketone bodies in the blood leads to ketonuria (above 20 mg/dl,
sometimes concentration of ketone bodies may rise up to 350 mg/dl; normal level of
ketone bodies is <2 mg/dl). The accumulation of ketone bodies reduces the buffer
capacity of the blood and causes acidosis (ketoacidosis). Thus, the accumulation
of ketone bodies in the blood is a consequence of metabolic deregulation and not
necessarily to meet energy demands.
Hyperlipidemia is associated with an increasing the blood VLDL and CM levels.
Insulin deficiency causes a decrease in the LP-lipase synthesis and activity and a drop
in the rate ofVLDL and CM utilization. Thus dietary fats arc not deposited in adipose
9.8. MetaDoi ic Changes in Diabetes Mellitus 529

tissue due to the reducing storage processes and low activity of LP-lipase, and enter
the liver, where they arc converted imo triacylglycerols, which are transported from
the liver as part of VLDL. Nonenzymatic glycosylation of apoproteins and receptors
disrupts the interaction of receptors with lipoproteins and reduces their entry into
cells, which also leads to hyperlipidemia.
Insulin deficiency results in the protein synthesis rale slowdown and acceleration
of body protein breakdown and in patients with poorly controlled diabetes can leads
to an increase in the blood amino acid concentration. Amino acids enter the liver and
undergo deamination. The resulting ammonia is included the ornithine cycle, which
leads to a rise in. urea production, and in severe insulin deficiency an increase in. blood
urea concentration and, accordingly, in the urine may be experienced. The nitrogen-
free glycogenic amino acid residues arc included in gluconeogenesis, which further
enhances hyperglycemia.
Polyuria and polydipsia arc common diabetes signsand symptoms. The capability
of kidney to excrete high concentration of glucose and ketone bodies is limited.
Excretion of large amounts of osmotic active molecules such as glucose and ketone
bodies requires much more water excretion. Patients with DM excrete urea 2—3 Limes
more than a healthy human and the loss of water is accompanied by excessive thirst.
For example, urine excretion in some cases reaches 8-9 liters per day, but more often
it docs not exceed 3-4 liters. The water loss causes constant thirst and an increase in
water consumption. In severe forms of diabetes dehydration can occur. As a result of
the excretion of large urine quantities, blood volume decreases: cells receive water
from the extracellular fluid; the extracellular fluid becomes hyperosmolar and «pump*
water out of the cells; external signsofdehydration develop — dry mucous membranes,
loose and wrinkled skin, sunken eyes, blood pressure decreases and oxygen supply to
the tissues reduces.
Acute complications of diabetes (comatose states) arc resulted from the changes
in carbohydrates, fats and proteins metabolism. Diabetic coma manifests as a sharp
impairment of all body functions, accompanied by loss of consciousness. Acidosis
and tissue dehydration are the main precursors of diabetic coma (Fig. 9.19). In
decompensation of diabetes, a disorder of water and electrolyte metabolism develops.
It is caused by hyperglycemia, accompanied by an increase in osmotic pressure in
the bloodstream. To maintain the osmolarity, a compensatory movement of the fluid
from the cells and the extracellular space into the bloodstream begins. This leads to
tissue loss of water and electrolytes, primarily Na'.. K , Cl . and HCOq ions. As a
result, severe cellular dehydration and a deficiency of intracellular ions (mainly KJ
develop, accompanied by general dehydration. This leads to a decrease in peripheral
blood circulation, a decrease in cerebral and renal blood How and hypoxia. A diabetic
coma develops slowly over several days, but can sometimes arise in a few hours. The
first signs may be nausea, vomiting, lethargy. Patient’s blood pressure is reduced.
1 n diabetes me Hilus comatose states can manifest in three main forms: kctoacidotic,
hyperosmolar and lactic acidotic.
Diabetic Ketoacidosis (DKA) may be caused by the cessation or reduction of
insulin administration, late diagnosis of the disease, infectious diseases, stress state.
The metabolism in DKA simulates that in starvation with marked insulin deficiency
but the intensity of lipolysis, gluconeogenesis and ketogenesis is very high in DKA,
530 Chapter 9. Molecular endocrinology

resulting in acidosis. polyuria, polydipsia. Hypcrglucoscmia (20-30 mmol/L).

caused by insulin deficiency, is accompanied by large losses of fluid and electrolytes,
dehydration and hyperosmolar plasma. The total concentration of ketone bodies
reaches 100 mg/dL and higher.

Fig. 9.19. Mechanisms ol development of diabetic soma. Metabolic changes of diabetic coma arise
from insulin deficiency with increased levels of counterregulatory hormones like catecholamines,
glucagon, cortisol and growth hormone. The changes of hormonal ration result in increased glucose
concentration because of inadequate glucose utilization by insulin sensitive tissues like muscles,
adipose tissue and liver; increased glycogenolysis; and increased gluconeogenesis by the liver from
amino acids, increased lipolysis leads to increased free fatty acids undergoing p-oxidation io generate
ketone bodies. High intensity of lipolysis, gluconeogenesis and ketogenesis leads to profound acid­
base and electrolyte disturbances. Osmotic diuresis results in loss of large amounts of water, sodium,
potassium, magnesium and phosphate
9.8. MetaDolic Changes in Diabetes Meiliius 531

In hyperosmolar coma the major criterion is extremely high blood glucose level
associated with markedly elevated scrum osmolarity (> 340 mOsm/L, if the patient
is comatose) with minimal or no ketoacidosis, polyuria* polydipsia, and always
manifested severe dehydration. It is associated predominantly with type 2 diabetes.
But* it is important to realize that any of these metabolic emergencies can occur in any
types of I) M, irrespective of the age or gender of the patient.
Hypotonia, a decrease in peripheral circulation and tissue hypoxia, that leads to
metabolism shifting towards anaerobic glycolysis, with increased the blood lactate
concentration (lactic acidosis), arc predominant in lactic acidosis.
Pure forms of the comatose states described arc practically not found. Their
development may be caused by various factors, for example, infectious diseases,
injuries, surgical interventions, toxic compounds, etc.
Late complications of diabetes niellitus are associated with long-term damage and
failure of various organ systems and a consequence of prolonged hyperglycemia and
often lead to early disability of patients. Late diabetic complications include a variety
of clinical pictures, mainly related to the involvement of the arterial wall both of large
vessels (macroangiopathy) and small vessels (microangiopathy), and of the peripheral
nervous system (neuropathy). Hyperglycemia causes damage to blood vessels and
impaired function of various tissues and organs.
The main mechanism of tissue damage in diabetes is glycosylation of proteins and
the associated dysfunction of tissue cells and changes in the rheological properties
of blood and hemodynamics (fluidity, viscosity). Some compounds normally
contain carbohydrate components (glycoproteins, proteoglycans, glycolipids). These
compounds arc synthesized by enzymatic reactions (enzymatic glycosylation).
However, non-enzy malic interaction of glucose aldehyde group with free amino
groups of proteins (non-enzymatic glycosylation) can also occur in. the human
body. In tissues of healthy people, this reaction proceeds slow and accelerates with
hyperglycemia.
One of the first signs of diabetes is a 2-3 times increase in glycosylated
hemoglobin. Throughout the lifetime of the erythrocytes* glucose freely penetrates
their membranes. Then in non-enzy malic reaction it irreversibly binds to hemoglobin,
mainly to 0-chains to produce a glycosylated hemoglobin HbA|r. Small amounts of
HbA can be also found in healthy people. In chronic hyperglycemia, the percentage
of HbAk in relation to the total amount of hemoglobin increases.
The degree of proteins glycosylation depends on the rale of protein turnover. As
proteins age they arc more likely to acquire molecular damage. Proteins with, slower
rates of turnover arc, therefore, al greater risk of suffering a deleterious modification.
Long-lived proteins accumulate more alterations and include extracellular proteins,
matrix, basement membranes and crystalline (lens of the eye). Thickening of the
basement membranes is one of the early and permanent signs of diabetes, manifested
as diabetic angiopathies.
Diabetic macroangiopathies represent the changes manifested in a decrease in
arterial elasticity, damage to large and medium vessels of the brain and heart, lower
extremities and include such com plications as coronary artery disease, cerebrovascular
disease, peripheral artery disease, non-sign ifleant carotid stenosis and poly vascular
532 Chapter 9. Molecular enaocrinoiogy

disease. They develop as a result of glycosylation of the extracellular matrix proteins,

collagen and elastin, with their decrease in the elasticity of blood vessels and impaired
blood circulation.
Diabetic microangiopathies can be due to damage of capillaries, small vessels
and include damage to eyes (retinopathy), to kidneys (nephropathy) and to nenes
(neuropathy) as well as diabetic foot disorders. Some late complications of diabetes
nicllitus (cataracts, retinopathy) can be caused by an increase in the rate of glucose
conversion to sorbitol. Sorbitol is not used in other metabolic pathways, and its rate
of diffusion from celts is small. In patients with diabetes, sorbitol accumulates in the
retina and lens of the eye, kidney glomerular cells, Schwann cells, and endothelium.
High sorbitol concentrations arc toxic to cells. Its accumulation in neurons leads to
osmotic pressure increase, cell and tissue swelling. The clouding of the lens (cataract)
can develop both due to the lens swelling and disruption of the crystalline structure
caused by the sorbitol accumulation, and due to the glycosylation of the crystalline
lens, with formed multi molecular aggregates that increase the lens refractive power.

Review Tests
1. Match the figure an d the letter. Dynamics of change:
A. In a healthy person after a meal-
B. Under the insulin action.
C. Under the glucagon action.
D. Ina patient with diabetes.
E. In a patient with an increase in. glucose tolerance.

2. Match the figure and the letter. Metabolic change:

A. A decrease in the blood amino acid concent ration.
B. Protein catabolism.
C. Hyperglycemia.
D. Azotemia.
E. Gluconeogenesis.
9.8. MetaDoi ic Changes in Diabetes Mellilus 533

[ insulin deficiency j

I
I

21
I
.31
I
Turea level in the urine

J. Match the figure and the letter. Metabolic change:

A. Glycogen catabolism.
B. Gluconeogenesis.
C. Decrease in glucose utilization.
D. Glycogen synthesis.
E. Glycolysis.
[ insulin deficiency

Hyperglycemia

4. Match the figure and the letter. Events:

A. Impaired formation of mature insulin.
B. Enhancing the catabolism of glycogen, fat, protein.
C. Violation of the pre pro insulin synthesis.
D. Hyperglycemia, azotemia, kclonemia.
E. Polyuria.

p-cell damage
I
GJ
I
(2.1
I
d
534 Chapter 9. Molecular eoaocrmoiogy

Situational problems
LA student has fasted for a day to lose weight. How did the carbohydrate
metabolism change when the absorptive state was switched to 24 hrs fasting? For
answer the question:
a) compare the changes in carbohydrate metabolism in the student's liver after
the last meal and by the end of the first day of fasting, and draw appropriate
charts;
b) list the peptide hormones with an increased level in the student’s blood
during the absorptive state and by the end of the first day of fasting, name the
site of their synthesis, secretion stimuli, target organs and draw the diagram
of mechanism of signal transduction of these hormones in the target cells;
c) name the regulatory enzymes of the metabolic pathways with increased
activity and amount under the action of these hormones.
2. A girl decided to lose weight and has been fasting for three days. What metabolic
changes occurred in the girl’s organism by the end of the 3-day fast? For answer
a) draw appropriate charts of metabolic pathways reflecting the changes in fat
metabolism in the liver and adipose tissue during the prolonged fasting;
b} name the hormones that accelerate these processes, and draw the charts of
signal transduction of these hormones to the target cells;
c) enumerate the regulatory enzymes with changed activity under the influence
of the mentioned hormones.
3. Tourists miscalculated food supplies and have been starving for 2 days before
reaching a settlement. How has their fuel metabolism changed in this case? For
answer:
a) explain how the concentration of glucose in the blood of tourists changed by
the end of the second day of fasting:
b) enumerate the metabolic pathways that maintain the blood glucose
concentration on the first day of fasting;
c) name the hormones that regulate the blood glucose levels during this period?
d) draw a chart of the signal transduction of these hormones.
4. Tourists were cast away on a deserted island and were starving for a week. None
of them developed a hypoglycemic coma. By chance, a captain of a cruise liner
came to their rescue and delivered them to a medical center on land where they
were examined. The blood glucose concentration in everyone did not fall below
65 mg/d L. Why was the blood glucose concentration of tourists within the lower
limit of the norm? To answer:
a) explain the molecular mechanisms that maintain the blood glucose levels
during long-term starvation;
b) draw charts for the metabolic pathways with increased activity under these
conditions; specify regulatory enzymes;
c) enumerate the hormones that stimulate these metabolic pathways;
d) present a diagram of signal transduction of these hormones on hepatocytes.
5. There is an expression; diabetes mellitus is ^hunger among abundance.* What
metabolic changes in diabetes confirm the validity of this statement? For
answer:
9.8. MetaDoi ic Changes in Diabetes Melliius 535

a) list the main causes of metabolic changes in IDDM;

b) enumerate the tissues with the main energy sources metabolism proceeding
according to this type of starvation;
c) name the metabolic pathways that are activated and inhibited in these tissues,
and explain why;
d) list the symptoms of diabetes mcllitus, that reflect such metabolic changes;
e) draw a diagram of one of the metabolic pathways that is activated in the liver
under these conditions and explain the consequences of such activation.
6 A40-vcar-old woman was taken to a hospital by ambulance. She exhibited tangled
consciousness, the smell ofacetone from the mouth, rapid breathing, xerostomia.
Screening showed that the blood glucose concentration is 18 m mol/L (324 mg/
dL); ketone bodies — 4.9 mmol/dL (norm 0.02-0.43). Urine contained ketone
bodies. Arterial pH is 7.3. Based on the studies conducted, diabetic ketoacidosis
was diagnosed. What metabolic changes could lead to the situation described
above? For answer:
a) suggest the type of diabetes and give the changes of hormonal status associated
with diabetes mcllitus;
b) list the main symptoms of the disease and explain their causes;
c) draw the charts of lipid metabolism pathways in adipose tissue and liver with
an acceleration leading to ketoacidosis;
d) name the immediate actions to improve the patient's condition.
7. A 35-year-old villager with IDDM was examined in the hospital and assured the
endocrinologist that he has kept strictly to the recommended diet during insulin
treatment. The screening showed that the blood glucose concentration was
6.2 mmol/L; HbAk level was 11.7% (norm 4—6%); no glucose or ketone bodies
were detected in the urine. After reviewing Lhc results of laboratory studies,
the attending physician was in doubt that the patient followed his nutritional
guidelines. Explain this doctor's Query. For answer:
a j name the reason for the blood HbA, increase;
lc
b} list the late complications of diabetes mcllitus and explain the molecular
mechanisms of their occurrence.
8. In patients with diabetes mcllitus type I, the biochemical disorders result from
changes in fuel metabolism . One of these signs is acidosis. Explain why such
patients have a deviation of blood pH from the norm? For this:
a) name the compounds with their accumulation causing acidosis, write their
structural formulas;
b) write the reactions of synthesis and oxidation of these molecules, name the
enzymes, coenzymes, reaction localization:
c) explain the origin of the precursor used in synthesis of these molecules, name
appropriate metabolic pathways;
d) specify the hormone that accelerates this precursor formation and provide
appropriate charts, starting from the hormone binding to adipocyte and
concluding with precursor formation, give an explanation to the charts.
9. A man in an unconscious state suspected to result from fasting ora diabetic coma
was taken to the emergency room of the nearest hospital. The patient’s breath
536 Chapter 9. Molecular eoaocrmoiogy

smells of acetone. Explain what needs io be done to determine the patient’s

diagnosis. For answer:
a) name the ketone bodies and conditions causing their increased level in.
blood;
b) explain which laboratory tests should be performed to diagnose the patient’s
condition;
c) name the symptoms characteristic of fasting and diabetes:
d) describe the similarities and differences in the metabolism of carbohydrates,
fatsand proteins for both conditions.
10. Two men with, hyperglycemia visited an endocrinologist. One patient was
diagnosed with I tscnko-Cushi ng’s disease; another was diagnosed with diabetes
mcllitus. Explain the differentiation in these disorders. What metabolic changes
will be characteristic in these patients? For answer:
a) compare the changes in carbohydrate metabolism lor both patients;
b) enumerate the metabolic changes for diabetes mcllitus;
c) explain which disorder causes ketoacidosis and why.
11. Apalicntcame toalhcrapist with complaintsofprogressive weakness, drowsiness,
dizziness. Symptoms were intensifying du ring fasting that allowed the doctor to
suggest hypoglycemia in the patient. Additional screening (glucose level was
less than 2.5 mmol/1. C-pcptide level significantly increased) confirmed the
initial assumption. The patient does not suffer from diabetes and docs not take
sugar-lowering drugs. What possible cause of the disease can be assumed in
the patient? What hormone secretion is increased in such patient? What is the
danger of hypoglycemia? What processes in the body prevents the hypoglycemia
even during fasting in healthy person?

9.9. ACTIONS OF HORMONES REGULATING SODIUM AND WATER

BALANCE
Normal cellular function requires maintaining water and electrolyte homeostasis
within very narrow limits. The main parameters of cell volume and osmolality (the
ratio of solutes in a solution to a volume of solvent in a solution=soluLe concentration
per litre of intracellular fluid) arc osmotic pressure, pH and the volume of intracellular
and extracellular fluid. Changes in these parameters can lead to changes in blood
pressure, acidosis or alkalosis, dehydration, and edema. Survival depends upon our
ability to compensate wide variations in intake and loss of both electrolyte and water by
activating homeostatic mechanisms designed to return water and solute concentrations
to normal as quickly as possible. Aniidiuretic hormone (ADH), aldosterone and atrial
natriuretic peptide (ANP) arc the main hormones that accomplish the water and
electrolyte balance regulation.
Antidiuretic hormone (ADH), also known as vasopressin, is a peptide containing
nine amino acids connected by one disulfide bridge. Ills synthesized as a pre hormone
in. the supraoptic and paraventricular nuclei, of hypothalamus. The gene for ADH also
encodes neurophy sin II, a carrier protein that transports ADH along axons of neurons.
9.9. Actions of Hormones Regulating Sodium and Water Balance 537

which terminate in the posterior pituitary where ADH is stored, and then secreted into
the bloodstream with appropriate stimulation.
The release of ADH from its storage vesicles in the neurohypophysis (posterior
pituitary; pars nervosa) is primarily caused by increase the plasma osmolality
(osmotically active solute concentration in millimolcs/kg of plasma waler) and
increase the osmotic pressure of the extracellular fluid (Fig. 9.20).

Urine ceil

Fig. 9.20. Secretion and action of ADH. ADH is produced Dy neurons in tne hypothalamus and stored
bound to its neurophysin (NPIIJ in tne posterior pituitary. ADH is synthesized and released in response
to increased sodium level and decreased plasma volume. ADH Dinos to cell membrane receptors,
stimulating tne activation of protein kinase A {PKA)_ PKA phosphorylates proteins that stimulate
synthesis of aquaporin, a protein that forms channels in the renai tubular membrane. Water, resorbed
through these channels from the urine, enters the blood
538 Chapter 9. Molecular endocrinology

The most important target cells for ADH arc the cells of the distaJ tubules and the
collecting tubules of the kidneys. The cells of these ducts arc relatively impermeable
to free water, and in. the absence of ADH, the urine is not concentrated and can be
excreted in quantities exceeding 20 liters per day (the norm is 1-1.5 liters per day).
The biological effects of ADH arc mediated through two types of receptors, V(
and V2.
The major action of vasopressin binding to V.-reccptor on renal tubular cells is to
promote waler reabsorption through the luminal membrane of the epithelial cells of
the cortical and medullary segments of the kidney collecting ducts ( Fig. 9.21).

Kinase A

I
Water
reabsorption

/ medullary principal cell

\inner
Fi^ 9.21 Mechanism of ADH action is through surface Vi (vascular srnoom muscle) and VE (kidney)
receptors. The v( receptor mediates me pressor actions of ADH, and the v? receptor mediates me
water conservation effects

The complex ADH — V.,-receptor causes activation of the adcnylyl cyclase., which
produces cAMP. cAMP activates protein kinase (PKA), which phosphorylates
proteins that stimulate the expression of the membrane protein gene, aquaporin-2.
Aquaporin-2 moves to the apical membrane, integrates into it and forms waler
channels through which water molecules diffuse freely into cells of renal tubules,
and then enter the interstitial space. As a result, water is reabsorbed from the renal
tubules.
Receptors V'^rc localized in smooth muscle membranes. The interaction of ADH
with the V]-receptor leads to the activation of phospholipase C, which hydrolyzes
phosphatidylinositol 4,5-bisphosphate forming diacyl glycerol (DAG) and inositol
1,4,5-trisphosphatc (IP,). IPjstimulates the release of Ca2k from the endoplasmic
reticulum and the vascular smooth muscle layer is contracted resulting in the volume
of the vascular bed decrease and the pressure rise.
9.9. Actions of Hormones Regulating Sodium and Water Balance 539

Diabetes insipidus is caused by a AD H deficiency with dysfunction of the posterior

lobe of the pituitary aland, as well as a hormonal signal transduction disturbance.
The main manifestation of diabetes insipidus is polyuria, i.e., excretion of large
quantities of low density urine (dilute urine). Mutations in the vasopressin receptor
gene and in the AQI^ gene lead to different types of nephrogenic diabetes insipidus, a
condition also associated with passing large amounts of urine and with dehydration.
An excessive secretion of ADH may occur following major trauma or surgery. This
is known as the syndrome of inappropriate antidiuretic hormone secretion and leads to
water retention.
.Aldosterone, the most potent mincralocorticosicroid, is synthesized in the cells of
the glomerular zone of the adrenal cortex from cholesterol. The synthesis and score lion
of aldosterone is stimulated with a low concentration of Na4, a high concentration of
K* and the renin-angiotensin system. The hormone diffuses from the blood into the
cytosol of target cells and interacts with its specific receptor, probably located both in
the cytosol and the nucleus (Fig. 9.22), and stimulates the gene transcription of several
proteins that arc essential to supporting the fiux of Na1. K . and Mg2, across target
cell membranes resulting then in the reabsorption of sodium ions and the excretion of
potassium ions.

LimEfi cd cwiicaJ
collecting dusr

Proteins in kidney tuDulat

Fig. 9.22. Aldosterone action in a kidney's cortical collecting duct celL
cells which synthesis is stimulated Dy aldosterone: 1 — components of Na4 channels that increase
me Na- resorption from the urine; 2 — enzymes of me IGA cycle that increase me capacity for ATP
production; 3 — Na*, K'-ATPases mat increase me rate at which Na* and K’ are exchanged Detween
me intracellular compartment and me blood
540 Chapter 9. Molecular endocrmoiogy

[n addition, aldosterone induces synthesis or proteins that increase the number

of pumps Na', K'-ATPasc and enzymes of the TCA cycle (e.g^ citrate synthase),
generating ATP molecules for active transport of ions. The net result of aldosterone
action is that the more. Na' is resorbed from the urine and more K1 is excreted.
One of the most important hormonal regulator that controls hemodynamic
stability by maintaining blood pressure, fluid volume, and sodium-potassium balance
is the renin-angiotensin-aldosterone system: (RAAS) (Fig. 9.23). Juxtaglomerular cells
of renal afferent arterioles that synthesize the proteolytic enzyme renin. A loss of both
fluid and blood, a decrease of NaCl concentration and hypotension in the afferent
arterioles of renal glomerulus, and sympathetic activation stimulate the release of ronin.
The glycoprotein angiotensinogen produced and secreted by the liver is hydrolyzed by
renin to form dccapcplide. angiotensin I. Angiotensin I acts as a substrate for ACE
(angiotensin-converting enzyme, carboxydipentydyl peptidase) which removes two
amino acids from the angiotensin I, primarily in the lungs, producing the octapcptide,
angiotensin II (All). Angiotensin II stimulates the inositol phosphate system and
increases the synthesis and secretion of aldosterone by the adrenal glomerulosa cells.
Iking also a potent vasoconstrictor, angiotensin II causes contraction, of the smooth,
muscle cells of the blood vessels, respectively, an increase of blood pressure and, in.
addition, stimulates thirst, which causes the patient to drink and thus to replete the
ECF volume.

Fig. 9.23 Reiiiit-angiotBiisiii-aldosterDiie system. Angiotensinogen is a glyco protein synthesized in

the liver. Renin is an enzyme produced in the juxtaglomerular apparatus of me kidney. Renin cleaves
angiotensin I from me angiotensinogen. Angiotensin-converting enzyme (ACE) produces angiotensin II

The renin-angiotcnsin-aldosteronc system provides the constancy of both ECF

volume and the blood volume , which, may decrease as a result of bleeding, profuse
vomiting, diarrhea and sweating that arc the states sig nailing release of renin (Fig. 9.24).
A decrease of the intravascular fluid volume reducing impulses from the baroreceptors
ofthe atria and arteries a Iso contributes to renin secretion. The formation ofangiotensin
II increases and. accordingly, the blood aldosterone concentration increases, causing
sodium ions retention.
9.9. Actions of Hormones Regulating Sodium and Water Balance 541

Na+ reabsorption—* Osmotic pressure—*■ ADH ——*■ Water reabsorption

K* excretion increase

Fig.9.24. Renin-angiotensin-aldosterone system. A decrease in fluid volume and a decrease in blood

pressure activate tne renin-angiotensin-aidosterone system. Angiotensin II causes vasoconstriction,
which is an emergency measure io maintain diood pressure. Aldosterone stimulates sodium retention,
as a result of wtiicn vasopressin is released ano water reabsorption is enhanced. Angiotensin II also
causes thirst, which contributes to an increase in body fluids

This acts as a signal for osmoreceptors of the hypothalamus and ADH secretion
from the nerve endings of the anterior pituitary, which stimulates the reabsorption
of water from the collecting tubules. Havi ng a potent direct vasoconstrictor action
on the resistance vessels of the body angiotensin 11 increases blood pressure and also
increases thirst. Excess water intake causes the body to retain waler to a greater extent
than normal. An increase of fluid volume and an increase in blood pressure lead to the
elimination of the stimulus that caused the activation of the renin-angiotensin system
and the secretion of aldosterone and, as a result, lead to the restoration of the blood
volume.
The reduction of perfusion pressure in the renal glomeruli can also occur due to
narrowing (stenosis) of the renal artery or nephrosclerosis. In this case, the entire
renin-angiotensin system is also included. But since the initial volume and blood
pressure are normal, the inclusion of the system leads to an increase in blood pressure
above the norm and the development of so-called renal hypertension.
542 Chapter 9. Molecular eoaocrmoiogy

[n an excessive increasing the extracellular lluid volume (for example, excessive

blood transfusion). RAAS activity is suppressed because the resulting increase in the
EC F volume causes an ^excessive si retch* signal in the renin-producing juxtaglomerular
cells ofthe kidney, and renin secretion decreases. As a consequence, angiotensin 11 and
aldosterone levels in the blood' also decreases, leading to vasodilation and increased
sodium and water excretion into the urine (as well as stool, sweat, and saliva), all of
which normalize the increasing the ECF volume.
llyperaldosteronism is a disease caused by the adrenal glands hypersecretion of
aldosterone. The causes of primary hyperaldosteronism (Conn syndrome) arc adrenal
adenoma or diffuse hypertrophy of the glomerular cells producing aldosterone. In
primary hv pcraldosteronism, an excess of aldosterone enhances sodium reabsorption
in the renal tubules. Increasing the concentration of Na' in plasma stimulates the
secretion ofantidiurctic hormone and water reabsorption by the kidneys. In addition,
the excretion of potassium, magnesium and proton ions is enhanced. As a result,
hypernatremia develops, causing, in particular, hypertension, hypervolemia and
oedema; hypokalacmia leading to muscle weakness, as well as magnesium deficiency
and metabolic alkalosis.
The cause of secondary hype raidosteronism is an increased level of renin and
anigiotensin 11, which stimulates the adrenal cortex and leads to excessive aldosterone
synthesis. Clinical symptoms are less pronounced than with primary aldosteronism.
Simultaneous determination of aldosterone concentration and renin activity in plasma
allows finally differentiating the primary (renin activity in plasma is reduced) and the
secondary hypcraldostcronism (renin activity in. plasma is increased).
Atrial natriuretic factor (ANP) is a 28-amino acid peptide with disulfide bridge
between two cysteine residues, produced, stored, and released mainly by the
cardiomyocytcs of the heart’s atrium. The main factors regulating the secretion of
ANP arc an increased blood volume and central venous pressure. Other stimuli
include elevated blood pressure, increased scrum osmolality, atrial tachycardia
(increased heart rale), and enhanced blood catecholamine levels. Glucocorticoids
also increase ANP synthesis through a regulating action on the ANP gene. The
kidneys, adrenal glands, and peripheral arteries arc the primary ANP target cells
(Fig. 9.25).
The plasma membrane ANP receptor is a catalytic receptor with guanylyl cyclase
activity. Its ligand-binding domain is located in the extracellular space. Conformation
of the intracellular domain of ANP receptor alters following ANP binding to the
receptor that has intrinsic guanyl ate cyclase activity increased and converted GTP
to the cyclic GMP. As a result of the ANP action, the formation and secretion of
renin and aldosterone is supressed. The several biological effects of ANP includes
inhibition of angiotensin 11. stimulation of aldosterone biosynthesis, stimulation of
the renal glome nil osa filtration rate (GFR), so as to promote Na/ natriums is and
vasorelaxation. The cumulative effect of ANP is an increase of the water and Na'
excretion and a decrease of blood pressure (Table 9.6).
9.10. Actions of Hormones tri at Affect Calcium and Phosphate levels 543

Fig. 925. Target tissues and actions of ANP

Table 9.6. Physiological actions of atrial natriuretic peptide

Kidney (a) Increased glomerular filtration rate (GFR) leading

to an increased Na' excretion in urina and thus a
correlated reduction in plasma volume
lb} Supression oi renin secretion

Smooth muscle ol arteries, Muscle relacsation weth reduction in blood pressure

capillaries, and veins

Adrenal zona cells Antagonism of angiotensin II stimulation of

algo sterone biosynthesis

Brain Complex set ol neuroendocrine and neurotransmitter

effects

ANP is usually considered as a physiological antagonist of angiotensin IL as it

causes dilation of blood vessels and loss of salt and water.

910. ACTIONS OF HORMONES THAT AFFECT CALCIUM

AND PHOSPHATE LEVELS
Calcium, as Lhc most abundant mineral in human body, is involved in many
physiological and pathological processes. The main functions ofcalcium arc as follows:
* calcium salts form the mineral matrix of bones:
* Ca2' ions are cofactors of many enzymes and non-enzy malic proteins:
544 Chapter 9. Molecular eoaocrmoiogy

► Ca2u ions in interaction with modulator proteins (c.g., calmodulin) act as an

intermediary in signal transduction regulating many biochemical processes and
physiological functions.
Adult body contains approximately I kg of calcium that forms two unequal funds.
One of these is bone calcium. Over 99% of total body calcium is found as calcium
hydroxyapatite (Ca1Q[POJt[OH|2) in bones and teeth, where it provides hard tissue
with its strength. Bone serves as a reservoir of calcium when deficiency exists and as its
store when the body is calcium replete.
The mineral components of the bone provide half of its mass: the other half is
formed by organic matrix. This matrix consists of organic components, mainly type I
collagen. Since the mineral part of the bone has a high density, it accounts for only a
quarter of the bone volume.
Other reserves of body calcium are Ca2' ions dissolved in fluids or combined with
proteins of fluids and tissues. There is a constant calcium exchange between these
reserves. Through, interacting with numerous proteins distributed in different cellular
compartments, intracellular calcium regulates a large amount of cell activities, such as
muscle.contraction, enzyme activation, cell differentiation and proliferation, immune
response, programmed cell death and neuronal activity. Regulation occurs by altering
Ca2 concentration that can be changed only by pumping from cell to intercellular fluid
or back, and from endoplasmic reticulum cisterns to cytosol or vice versa, etc. The
calcium concentration inside the cells depends on its concentration in the extracellular
fluid. In healthy people, the extracellular calcium concentrations are maintained
within an exquisitely narrow range because of the role it plays in cellular functions. I n
blood, total calcium concentration is normally 2.12-2—2.6 mmol/L (9-11 mg/dL).
and is a thousand limes less in the intracellular fluid. Calcium exists in the circulation
in three forms. The ionized Ca2 is the most important and physiologically active
form (50% of total calcium). The majority of the remaining calcium is protein bound,
mainly to negatively charged albumin (40%), and the rest is complexed to substances
such as citrate and phosphate (10%-).
The serum Ca2' concentration is precisely controlled since calcium ions arc
involved in muscle contraction, increase cell membrane permeability to potassium
ions, affect cell sodium conductivity and ion pumps, promote hormone secretion and
the blood coagulation cascade, as well as act as the essential mediators in intracellular
signaling. A change of only 1% in it drives the homeostatic mechanisms that restore
balance.
Ca2' is usually absorbed in the intestines accompanied by phosphates, and
deposition and mobilization of calciu m in bones always occurs in conjunction with
phosphates. Phosphates is required lor high-energy phosphate bond of ATP and other
metabolites. It. is a component of nucleic acids and second messengers (c.g., cyclic
AMP and IP-,), and as the addend that increases or decreases enzymatic activities or
guides protein-protein interactions. About 90% of the 500 to 800 g of phosphates in the
adult human is deposited in the skeleton. Much of the remainder is incorporated into
organic phosphates distributed throughout soft tissues in the form of phospholipids,
nucleic acids, and soluble metabolites. Daily intake of phosphates is in the range of
9.10. Actions of Hormones tri at Affect Calcium and Pnospnate levets 545

1,000 to 1,500 mg, mainly in dairy' products. Blood inorganic phosphate level is about
3.5 mg/dL. About 55% is present as Tree ions, about 35% is complexed with calcium
or other cations, and 10% is protein-bound.
Parathyroid hormone, cafcitriol and calcitonin are the main regulators of Ca2 and
phosphate exchange.
Parathyroid hormone is synthesized in the parathyroid glands in the form of
preprohornionc that is converted to mature 84-amino acid, single-chain peptide
hormone (PTH) by partial proteolysis. PTH is secreted in. response to a decrease in
extracellular ionized calcium or an increase in serum phosphate concentration. The
hormone causes the concentration of free scrum Ca2 to increase to a level just above
the normal range (above a critical set point). The latter acts as a * negative feedback*
signal to the chief cells of the parathyroid gland, inhibiting fun her release of PTH
until the free Ca2' level in the blood again falls below a specific threshold or set point
(Fig. 9.26). Aller secretion into the blood, intact PTH is inactivated by proteolysis.

Parathyroid
gland

PTH

Fig. 9.26. Effects ol PTH. • 1: PTH stimulates the mobilization of Ca?i from bone: (2) PTH stimulates the
reabsorption of Ca?* in the distal tubules of the kidneys: (3) PTH activates the formation Gtl.SSfOHjPg
in the kidneys, which leads to stimulation of the absorption of Ca3- in the intestine: {4) increased
concentration of Ca74 ions inhibits the secretion of parathyroid hormone

The main target organs for the hormone arc bones and kidneys. The hormone
initiates a cascade of events associated with osteoblast adenylate cyclase that stimulate
the metabolic activity of osteoclasts. Ca2’, is mobilized front the bone and phosphate
enters the blood. In the distal tubules of the kidneys Ca2', reabsorption increases and
phosphate reabsorption decreases, resulting in the restoration of normal calcium ion
levels in the extracellular fluid.
546 Chapter 9. Molecular endocrinology

Calcitriol, like other steroid hormones, is synthesized from cholesterol. The

ini mediate precursor of calcitriol is cholccalcifcrol (vitamin D3). A small amount
of vitamin D3 is presents in food, but most of the vitamin used in the synthesis of
calcitriol is synthesised in the skin from 7-dchydrocholcsterol by a non-cnzymatic
reaction under ultraviolet light (Fig. 9.27). Vitamin D3 is transported throughout the
blood and is taken by the liver, where it is oxidized to form 25-hydroxycholecalci feral
(25OH-Dj) by the same P450 mitochondrial enzyme that oxidizes cholesterol on
carbons 26 and 27 in the formation of bile acids. 25OH-D3 has high affinity for the
vitamin. D-binding protein and is the major circulating form of vitamin D. It has little
biological activity. In the proximal tubules of the kidney, a second hydroxyl group is
added at carbon I by the enzyme 1-hydroxylase. This hydroxylation in the kidneys
is the rate-limiting step and yields calcitriol l,25(OH),Dr the most active form of
vitamin D v The enzyme of this reaction is activated by a "low blood Ca2 concentration
and is stimulated by parathyroid hormone.

JlT I H
Or
I
J—t
r'A;
p (7-ctetiydfDchoie&iaror|
ho!^3jx

Skirt J-

(axrtecaiLil&.'aliviTa.-wi Pg))
"r

Liver
fgl-hydroajf' caJcjierof (25 OHDj ]

KiHrtEy

Fig WCalcitriol synthesis. {1,25-{1r(OH)?D2) is produced from 7-deftydrochoiesterol, derived from

cnolesteroL in me skin, UVIiant produces cnolecaiciferoi which is hydroxylated at the 25-position in
the 1-position in the kidney to form the active hormone
9.11. Disorders of Calcium Metabolism 547

Increasing the calcilriol concentration, on the contrary,, inhibits the synthesis of

kidney hydroxylase, stipressing the hormone formation. Calcilriol circulates in blood
bound to a carrier protein and then diffuses passively through the plasma membrane
of target tissues. In the intestine bone, and kidney the calcilriol then moves into the
nucleus and binds to specific receptors. This complex activates genes that encode
proteins mediating the calcilriol action. In the intestinal mucosa cell, for example,
transcription of genes encoding calcium-Iransporting proteins is activated. These
proteins arc capable of carrying Ca2' (and phosphate) absorbed from the gut lumen
across the cell, making it available for eventual passage into the circulation. Calcilriol
has an effect on the small intestine, kidneysand bones. Like other steroid hormones,
calcilriol binds to the intracellular receptor of the target cell. A hormone-receptor
complex formed interacts with chromatin and induces the transcription of structural
genes, resulting in the proteins synthesis that mediate the calcilriol action. For
example, in intestinal cells, calcilriol induces the synthesis of Ca2'-transfer proteins,
which ensure the absorption of calcium and phosphate ions from the intestinal cavity
into the intestinal epithelial cell and further transport from the cell to the blood, due
to which the calcium ions concentration in the extracellular fluid is maintained at the
level required for mineralization of the organic matrix of bone tissue. In the kidney,
calcilriol stimulates the reabsorption of calcium and phosphate ions. With a lack of
calcilriol, the formation of amorphous calcium phosphate and hydroxyapatite crystals
in the organic matrix of bone tissue is impaired, leading to the development of rickets
and osteomalacia. Il was also found that al a lowr calcium ions concentration, calcilriol
promotes the calcium mobilization from bone tissue.
Calcitonin is a linear potypepii.de consisting of 32 amino acid residues with one
disulfide bond. The hormone is produced by parafollicular K-cctlsof the thyroid gland
or C-cells of the parathyroid glands in the form of a preprohormone. The regulation
of calcitonin secretion depends upon changes in free calcium levels in the blood:
calcitonin secretion increases with increasing blood Ca2' concentration, and decreases
with decreasing blood Ca2' concentration.
The hormone acts on osteoclastic cells in bone (bone-resorbing cells) to suppress
their osteolytic activity. Calcitonin is a hypocalcemic hormone that acts as an antagonist
to PTH. It inhibits the release of Ca2' and phosphates from the bone, also suppresses
tubular reabsorption of calcium ions in the kidneys, thereby stimulating excretion of
Ca2 and phosphates by the kidneys in the urine, causing a fall in scrum Ca2 and
phosphates levels. The rate of calcitonin secretion in women is highly dependent on
the estrogen level. With a lack of estrogen calcitonin secretion is reduced. This causes
the acceleration of the mobilization of calcium from bone tissue, that leads to the
development of osteoporosis.

911 DISORDERS OF CALCIUM METABOLISM

When the blood calcium concentration is less than 9 mg/dL or more than 11 mg/dl
these states indicates respectively hypocalcemia or hypercalcemia. Changes in the blood
calcium level affect the calcium concentration inside the cells, that leads to a change in
the excitability threshold of nerve and muscle cells, impaired calcium pump function,
reduced enzyme activity and impaired hormonal regulation of metabolism. Moderate
548 Chapter 9. Molecular eoaocrmoiogy

to severe hypocalcemia is associated with paresthesias, usually of the fingers, toes,

and cireumoral regions, and is caused by increased neuromuscular irritability. Severe
hypocalcemia can induce seizures, carpopedal spasm, bronchospasm, laryngospasm.
Mild hypercalcemia (up to 11—11.5 mg/dL) is usually asymptomatic. More severe
hypercalcemia (>12—13 mg/dL) may result in gastrointestinal symptoms (nausea,
anorexia, constipation, or pancreatitis) and can cause neurological symptoms, such,
as depression, memory loss, and irritability. Severe cases can. cause lethargy, stupor,
or coma.
Hyperparathyroidism means excessive secretion of parathyroid hormone resulting
from a parathyroid tumor, diffuse gland hyperplasia and parathyroid carcinoma
(primary hyperparathyroidism) and leads to increased calcium and phosphate
mobilization from the bone, increased calcium reabsorption, and phosphate
elimination in the kidneys. As a consequence, hypercalcemia occurs, which can
lead to a decrease in neuromuscular excitability and muscular hypotension. Patients
develop general and muscular weakness, fatigue and pain in certain groups of
muscles, and the risk of fractures of the spine. femur and forearm bones increases.
An increase in the concentration of phosphate and calcium ions in the renal tubules
can cause the formation of stones in the kidneys and leads to liypcrphosphaturia and
hy pophosphatem ia.
Hypoparathyroidism caused by parathyroid insufficiency leads to hypocalcemia.
Lowering the blood calcium ions concentration can cause neurological, ophthalmic
and cardiovascular disorders, as well as damage to the connective tissue. A patient with
hypoparathyroidism has an. increase in neuromuscular conduction, attacks of tonic
convulsions, convulsions of respiratory muscles and diaphragm, and laryngism.
Rickets is childhood skeletal disorder associated with insufficient mineralization of
bone tissue. Impaired bone mineralization is a consequence of calcium deficiency and
may be due to the following reasons:
► lack of vitamin calcium, or phosphate in the diet:
► impaired absorption of vitamin Ik in the small intestine:
* reduced synthesis ofcalcitriol precursors due to insufficient sun exposure:
► de fee L of I a- hyd roxyl asc;
► defect ofcalcitriol receptors in target cells.
All this leads to a. decrease in calcium absorption in the intestine and a reduction
of its blood concentration, stimulation of the secretion of parathyroid hormone and,
as a result, the mobilization of calcium ions from the bone. Symptoms of rickets can
involve multiple skeletal deformities (an oddly shaped skull, bowed legsand thickened
ankles and wrists, a protruding breastbone, a curved spine, pelvic deformities), stunted
growth and short stature, the abdomen growth, teeth deformities (c.g.? delayed tooth
formation, holes in the enamel, abscesses, defects in the tooth structure, an increased
number of cavities). Treatment for rickets focuses on. replacing the missing vitamin or
mineral in the body and sufficient insolation.
In the adult humans vitamin D,-deficiency results in osteomalacia. Mineralization
of osteoclasts to form bone is impaired, and such undermine ralizcd bone is structurally
weak. Since osteomalacia occurs after growth and development of the skeleton arc
complete its main symptoms arc muscular weakness and bone pain, with little bone
deformity.
9.11. Disorders of Calcium Metabolism 549

Review Tests
Match the number and the letter.
1. Regulation of hormone secretion. Molecule:
A. Natl.
B. Aquaporin.
C. Ncurophysin.
D. ADH.
E. V, receptor.
2. The mechanism of aldosterone action.
Induced proteins:
A. Proteins forming the Na' and K‘
channels.
B. Na', K-ATP-ase.
C. ATP synthase.
D. Citrate lyase.
E. Citrate synthase.

3. Recovery of fluid volume during blood loss. Molecules:

A. Aldosterone.
B. ADH
C. Angiotensin II.
D. Renin.
E. Angiotensinogcn.
 Hunger

4. The ANP effects in target cells. .Action hormone:

A. RclaAalionofblood vessels.
B. Inhibition of hormone secretion.
C. Vasoconstriction.
D. Inhibition of enzyme secretion.
E. Decrease in ihc activity of guanylatc cyclase.
9.11. Disorders of Calcium Metabolism 551

Situational Problems
1. On a hot day a tourist in the wilderness could not find a source of drinking waler
for a long time. Finally, he reached a village and quenched his thirst. How will
his water and salt balance change after that? For answer:
a) name the hormones that regulate the water and salt balance;
b) draw a chart explaining the occurrence of thirst;
c) using the chart, compare the state of the tourist’s water and salt metabolism
before he drank water and after.
2. A 4-month-old child has signs of rickets. Digestive disorders were not noted.
The child has had adequate sun exposure, he has been receiving vitamin D-, for
2 months, but the manifestations of rickets did not diminish. Why did the doctor
prescribe calcitriol to treat rickets in this child? For answer:
a) draw a chan of hormone synthesis derived from vitamin D5 and list the
possible causes of rickets in children;
b) enumerate the target organs of the hormone and draw a chart of the hormonal
signal transduction;
c) name the cause of rickets in this child and explain the doctors prescription.
3. A 45-year-old woman was admitted to hospital because of frequent headaches,
muscle weakness, increased thirst. Increased blood pressure, impaired blood
electrolyte composition and significantly reduced renin levels were determined
during the examination. A hormone-producing tumor in the right adrenal gland
was detected during a computed tomography scan, allowing to diagnose pri mar}'
hype raldosteron ism (Conn’s syndrome). What are the causes of hypertension in
this patient? For answer;
al list the biological functions of the main adrenal cortex hormones, name the
precursor for their synthesis;
b) name the main mineralocorticoid, the site of its synthesis and the target cell;
c) draw a diagram of the hormonal signal transduction to the target cells;
d) present a chart explaining the regulation of the hormone’s synthesis and
secretion;
c) explain the consequences of the hormone hyperproduction.
4. A 39-ycar-old patient was presented writh persistent arterial hypertension
(200/120 mm Hgh which was not eliminated by conventional antihypertensive
drugs. An ultrasound of the renal arteries revealed signs of stenosis of the right
renal artery. Why docs renal artery’ stenosis lead to an increase in blood pressure.
For explanation:
a) present a chart of water and salt balance regulation, indicate the main stimuli
of system activation and the final effects;
b) name the compound, with its increased synthesis caused by a drop in renal
arteries perfusion pressure;
c) describe the consequences arising from an increase in the blood concentration
of this substance, using the scheme presented in paragraph a).
5. A 23-ycar-old man was admitted to a hospital fora planned surgical operation.
When performing the surgical operation to remove a tumor from the upper
section of the anterior pituitary, an isthmus of the posterior pituitary was
552 Chapter 9. Molecular eoaocrmoiogy

affected. In die postoperative period, in the patient polyuria developed. How to

explain die patient symptom? For answer:
a) name die hormones synthesized in the hypothalamus and secreted from die
posterior lobe of the pituitary;
b) draw- a schematic mechanism of this hormone signal transduction;
c) name the hormone effects.
6. Increasing the aldosterone secretion during prolonged muscle activity in hot
weather allows you to compensate for the sodium loss in. sweat and remove
the accumulated excess of potassium. Explain these biological effects of the
hormone during exercise. For answer:
a) draw the structure of the hormone, name the precursor for its synthesis, site
of synthesis, signals for secretion, target cells;
b) draw a diagram of the hormonal signal transduction starting with hormone
interaction with the cell receptor:
c) explain the role of the hormone in the regulation of gene transcription and
translation;
d) describe the role of proteins that have their synthesis regulated by aldosterone.
7. A 45-year-old man was admitted to a hospital with complaints of depression,,
lack of appetite, general and muscular weakness. The examination revealed a
parathyroid adenoma. The blood parathyroid hormone level was increased.
What caused in the patient symptoms? For answer:
a) indicate the chemical structure of parathyroid hormone, name the stimuli of
its secretion, target organs:
b) draw the scheme of hormonal signal transduction mechanism;
c) describe the biological effects of parathyroid hormone excessive secretion
and explain these symptoms development.
8. A 55-ycar-old man presented to the hospital with complaints of general and
muscular weakness, hypertension, headaches, thirst, edema. When examining
the patient’s blood, hypokalemia and hypernatremia were delected. Computed
tomography revealed a tumor in the adrenal cortex. What disease can correspond
to these data? Explain the cause of the symptoms. For answer:
a) name the hormone, which synthesis and secretion increases in this patient,
the site of its synthesis, the stimuli of secretion and the target organs:
b) draw a schematic mechanism of hormonal signal transduction;
c) using the scheme, describe the biological effects of the hormone and the
mechanism of the patient's symptoms development.
9. A child receiving adequate nutrition and vitamin D1 shows signs of rickets.
The blood calcium concentration is at the lower limit of normal. What are the
possible causes of rickets in this child? For answer:
a> describe the symptoms of rickets;
b) name the hormones that regulate the calcium ions exchange in the body, and
indicate their biological effects:
c) provide a scheme for the hormone synthesis from vitamin Dnand indicate the
possible causes of ricke ts.
9.11. Disorders of Calcium Metabolism 553

IO. A 55-yca.r-old woman presented to the hospital with complaints of muscle

weakness, fatigue, night cramps, pain in the lumbar region. When fluoroscopy
revealed a decrease in bone density, a violation of bone structure. The patient
was diagnosed with osteoporosis ^porous bones*). Osteoporosis is a common
cause of fractures of the spine, femur and forearm bones. The doctor prescribed
treat ment with calcitriol and calcium supplements. Calcitriol receptors exist in
two allelic variants U and b. Studies have shown that dominant homozygotes
BB are more susceptible to osteoporosis and this patient has this genotype.
What arc the other causes of osteoporosis and what is the mechanism of the
therapeutic e fleet of calcitriol? For answer:
a) indicate the target organs of calcitriol;
b) write a scheme for the calcitriol synthesis;
c) describe the mechanism of calcitriol action on target cells:
d) name the main causes of osteoporosis;
c) explain why the use of calcitriol and calcium preparations improves the
condition of patients with osteoporosis.
 Chapter 10
TISSUE METABOLISM.
LIVER DETOXIFICATION FUNCTION

10.1. Mechanism of Toxic Compounds Detoxification

10.2. Putrefaction in Large Intestine and Detoxification of Formed Products
10.3. Biotransformation of Medicines
10.4. Metabolism and Detoxification of Ethanol
10.5. Chemical Carcinogenesis

10.1 MECHANISM OF TOXIC COMPOUNDS DETOXIFICATION

Xenobiotics arc compounds that have no nutrient value (cannot be used for energy
needs by the body) and potentially toxic. They arc present as natural components
of foods or they may be introduced into foods as additives or through processing.
Pharmacologic and recreational drugs arc also xcnobiolic compounds.
The liver is the principal organ in the body for the degradation of these
com poll nds. Potentially toxic agents absorbed from the gut or delivered to the liver
by the hepatic artery: NH,, products of amino acid p lie refaction in the intestine,
peptide and steroid hormones, catecholamines, and heme degradation products.
Pharmacological agents performed their functions have to be removed from the
body in invariable or modified form. Within the liver, these com pounds a re properly
metabolized, as well as potentially harmful substances arc detoxified and prepared
for subsequent excretion into the urine or feces. Among other functions, the liver
is equipped with a broad spectrum of detoxifying mechanisms. The sequential
transport steps carried out by such as endosomes, mitochondria, lysosomes, as
well as the nucleus include (I) uptake, (2) intracellular binding and sequestration,
(3) metabolism. (4) sinusoidal secretion, and (5) biliary excretion. The rate of
hepatobiliary transport is determined, in part, by the rate of activity of each of
these steps.
Detoxification stages arc divided into two phases. Because many of xenobiotics
arc lipophilic, they arc oxidized, hydroxylated, or hydrolyzed by enzymes in phase 1
reactions. Phase I reactions introduce or expose hydroxyl groups or other reactive sites
that can be used for conjugation reactions (the phase H reactions}.
The conjugation reactions add a nega Lively charged group such as glycine,
glucuronatc, sulfate, etc., into the molecule. Many xcnobiolic compounds will be
transformed through several different pathways (Fig. 10.1K
10.1. Mechanism of Toxic Compounds Detoxification 555

Ccqugahon

rPoiarity
iSzkitnIrty in'water

Fig. 101. General scheme ot xenoDiobc detoxification

First phase of detoxification

Many xcnobiotic compounds contain aromatic rings (such as benzopyrene in
tobacco smoke) or heterocyclic ring structures (such as the nitrogcn-containing
rings of nicotine or pyridoxine.) that arc unable to degrade or recycle into useful
components. These structures arc hydrophobic, causing the molecules to be retained
in cell membranes, and tissues rich with lipids such as nervous and adipose, unless
they are sequestered by the liver. kidneyT or intestine for biotransformation reactions.
In this phase, they undergo hydroxylation, sulfation, deamination, hydrolyzis, etc.
The cytochrome P450-dependent monooxygenase enzymes arc determinants
in oxidative, pcroxidalivc, and reductive degradation of exogenous (chemicals,
carcinogens, and pollutants, etc.) a nd endogenous (steroids, prostaglandins, retinoids,
etc J substances. The key enzymatic constituents of this system arc the flavoprotein
NADPH-cytochrome P450 oxidorcductase (FAD and FMN arc coenzymes} and
cytochrome P450(hcmoprotcin)(Fig. 10.2). The latter is the terminal eleciron acceptor
and substrate-binding site of the microsomal mixed-function oxidase complex, a very
versatile catalytic system. The major role of the cytochrome P450 enzymes is to oxidize
substratesand introduce oxygen to the structure.
The cytochrome P450(CYP450 ) enzyme family contains al least 1.00 to 150 different
isozymes with at least 40% sequence homology. These isozymes have different but
overlapping specificities. The human enzymes arc generally divided into six major
subfamilies, and each of these is further subdivided. For exam pic, the CYP3A4 isoform
accounts for 60% of CYP450 enzymes in the liver and 70% of cytochrome enzymes
in gut wall entcrocytes. Il metabolizes the greatest number of drugs in humans. The
concomitant ingestion of two CYP3A4 substrates could potentially induce competition
for the binding site, which, in turn, could alter the blood levels of these two agents.
The drug with the highest affinity for the enzyme would be preferentially metabolized,
whereas the metabolism (and degradation) of the other drug would be reduced!.
556 Chapter IQ. Tissue metabolism. Liver detoxification function

isadfh NADP^.H*

Cytadhrarre Cytochrwne
P450 reductase P45O

Fig, 1OJL General slructure of the P45Q enzymes. 0? Di nets to the P450 Fe-heme in the active site and
is activated to a reactive form by accepting electrons. The electrons are donated Dy the cytochrome
P450 reductase, whicn contains an FAD together with an FMN or (Fe-S)-center to facilitate the transfer
of single electrons from NADPH to O?. For example, for CYP2E1 (CYP2E1 is also referred to as me
microsomal ethanol oxidizing system, MEOS), RH is ethanol (CH3CHj£)H}, and ROH is acetaldehyde
(CH3COH)

The latter drug’s concentration in the blood would then rise. Moreover., many
substances or drugs impair or inhibit the activity of the CYP3A4 enzyme, thereby
impairing the body's ability to metabolize a drug.
The cholesterol-lowering agents known as the statins CH MG-CoA reductase
inhibitors) require CYP3A4 for degradation. Appropriate drug treatment and dosing
take into account the normal deg rad alive pathway of the drug. However, grapefruit
juice is a potent inhibitor of CYP3A4-mcdiaicd drug metabolism. Evidence suggests
that if a statin is regularly taken with grape fruit juice, its level in the blood may increase
as much as IS-fold, This marked increase in plasma concent ration could increase the
muscle and liver toxicity of the statin in question. Because side effects of the statins
appear to be dose-related.
The cytochrome P450 isozymes all have certain features in common. The
term «P450* is derived from the spectrophotometric peak al the wavelength of the
absorption maximum of the enzyme (450 nm) when it is in the reduced state and
complexed with carbon monoxide. Most CYPs require a protein partner to deliver one
or more electrons to reduce the iron (and eventually molecular oxygen).
All of them:
I ) contain cytochrome P450, oxidize the substrate, and reduce oxygen;
2) have a flav in-containing reductase subunit (hat uses NAD PEL and not NADH,
as a substrate;
3) found in the smooth endoplasmic reticulum and arc referred to as microsomal
enzymes (lor example, CYP2E1 is also referred to as the microsomal ethanol
oxidizing system, MEOS);
10.1. Mechanism of Toxic Compounds Detoxification 557

4) bound to die lipid portion of the membrane, probably to phosphatidylcholine;

5) inducible by the presence oft heir own best substrate and somewhat less inducible
by die substrates for other P450 isozymes;
6) generate a reactive free radical compound as an. intermediate in the reaction.
Cytochrome P450 binds lipophilic compound (RH) and oxygen in active
site; oxygen binds two electrons and converted to O2 . Electron and proton donor
is NADPH + Hl oxidized by cytochrome P450 reductase. O1 binds protons with
water formation: O- + 2H* -- H3O. The second oxygen atom is incorporated into the
hydroxyl group of compound R-OH.
The summary reaction:

RH+ O2+[NADPH+H1 -ROH + H.O + NADP'.

Hydroxylation enhances the solubility of the hydrophobic compou nd and decreases

its toxicity, as well as facilitates its further inactivation and excretion.

Second phase of metabolism — conjugation reactions prepare

xenobiotics for excretion
Conjugation is the other groups and molecules binding to functional groups formed
in phase 1 or been present in xenobiotics catalyzed by enzymes, which arc transferases.
These derivatives arc conjugated with molecules such as glucuronic acid, sulfate,
glycine, acetate, methyl group or glutathione. This renders them even more water-
soluble, and less toxic, and they arc eventually excreted in the urine or bile.
Glucuronidation. UDP-glucuronic acid is the glucuronyl donor, and a variety of
glucuronyl transferases, present in both the endoplasmic reticulum and cytosol, arc
the catalysts (Fig. 10.3).

u DP-glucuronic Acid Pnend Ether glucuronide (Phyryl glucuronide}

U DP-glucuronic Ackl Benzoic Acid Ester glucuronide (Benzoyl glucuronide |

Fig. 10.3. Phenol and benzoic acid detoxification by glucuronidation

558 Chapter IQ. Tissue metabolism. Liver detoxification function

The reaction in general:

ROH + UDP-C4H9O4= RO-CfiH,Ofi + UDP.

Sonic alcohols, arylamines, and phenols arc sulfated. The sulfate donor in these
and other biologic sulfation reactions (e.g.s sulfation of steroids, glycosaminoglycans,
glycolipids, and glycoproteins) is adenosine 3 -phosphate-5’-phosphosulfate (PAPS):
this compound is called «active sulfate* (Fig. 10.4):

Fig. 10 4. The conjugalion reaction with sulfate

Conjugation with glutathione. Glutathione {y-glutamyl-cystcinyl-glycinc) is a

tri peptide consisting of glutamic acid, cysteine, and glycine (Fig. 10.5 A). Glutathione
is commonly abbreviated GSH (because of the sulfhydryl group of its cysteine, which
is the business pan of the molecule). A number of potentially toxic electrophilic
xcnobiotics (such as certain carcinogens) arc conjugated to the nucleophilic GSH in
reactions that can be represented as follows (Fig. I0.5 B), where R = an electrophilic
xcnobiotic:

R + GSH - R-S-G.

The enzymes catalyzing these reactions arc called glutathione S-transferases and
varieties of them arc present in high amounts in liver cytosol and in lower amounts
in other human tissues. If the potentially toxic xcnobiotics were not conjugated to
GSH, they would be free to combine covalently with DMA, RNA, or cell protein
and could thus lead to serious cell damage. GSH is, therefore, an important defense
mechanism against certain toxic compounds, such as some drugs and carcinogens.
If the levels of GSH in a tissue such as liver arc lowered, then that tissue can be
shown to be more susccpiiblc to injury by various chemicals that would normally be
conjugated to GSH.
10.1. Mechanism of Toxic Compounds Detoxification 559

A
N CHj COO"
H

Y -gkJtamyt-s-y&tejny-glycine
Ghjlalhione

Dibromomelf^ane

*GSH

Conjugation reaction of some toxic compounds with glutathione: A — glutainione structure:

Fig. 10.5.
B — exam pies of conjugation witn GSH

Glutathione conjugates arc subjected to Further metabolism before excretion.

The glutamyl and give idyl groups belonging to glutathione arc removed by specific
enzymes, and an acetyl group (donated by acetyl-Co A) is added to the amino group
of the remaining cysteinyl moiety. The resulting compound is a mercapturic acid, a
conjugate of L-acctylcystcinc, which is then excreted in the urine. Glutathione has
other important functions in human, cells apart From its role in xenobiotic metabolism.
I . Il participates in the decomposition of potentially toxic hydrogen peroxide in the
reaction catalyzed by glutathione peroxidase.
2. It is an important intracellular reductant, helping to maintain essential HS-
groups of enzymes in their reduced state and its involvement in the hemolytic
anemia caused by the deficiency of glucose-6-phosphate dehydrogenase is
discussed in Ch. 6.4.
J. A metabolic cycle involving GSH as a carrier has been implicated in the transport
of certain amino acids across membranes in the kidney (Ch. 8.1).
560 Chapter 10. Tissue metabolism. Liver detoxification function

102. PUTREFACTION IN LARGE INTESTINE AND DETOXIFICATION

OF FORMED PRODUCTS
Most ingested food proteins arc digested to corresponding amino acids. which
arc absorbed from the small intestine. The residue (undigested proteins, peptides and
amino acids) passes into the large intestine. They undergo putrefaction by bacterial
enzymes (E — bacterial enzymes) of colon producing various gases, such as COV
methane (CHJ, hydrogen, and hydrogen sulfide, as well as solid compounds. Some
of these compounds arc potentially toxic for the human body.
Decarboxylation of some amino acids by intestinal bacteria produces toxic amines
(corpse poisons): cadaverine from lysine, putrescine from ornithine:

H2N — (CH2)4 — C— COOH H2N —(CHds— nh2

1 E
Lysine nh2 Cadaverine

H -CO2
h2n — — C —COOH H2N—(CH2)s— nh2
! E
Omitine nh2 Pulrescine

The amino acid tryptophan undergoes a scries of reaction to form skatole and
indole:

COOH

Tryptophan Indole
Phenylalanine and tyrosine arc converted to cresol and phenol:

Phenyalanine Cresol Phenol

The sulfur-containing amino acid cysteine undergoes a scries of transformations
to form mercaptans such as ethyl and methyl mercaptan, as well as H.,S. The large
intestine is the source ofconsiderable quantities ofammonia produced by deamination.
The largest part of these products arc eliminated with feces from human body, but
some amount of products of bacterial activity arc absorbed into the portal circulation
and rapidly removed from the blood by the liver where they arc inactivated under
normal conditions.
10.2. Putrefaction in Large intestine and Detoxification of Formed Products 561

Many toxic compounds arc detoxified by oxidatKm-mfacrion reacfioHi, by oxidases,

mono and dioxygenases (hydroxylation). Most oxidation reactions arc catalyzed by
cytochrome P450 systems (sec above):

OH
4-1/2^2

Monooxygenase
(hydroxylase) H

Indole Indoxyl

Co7jjtfgofe/o/7w<j//ow. The most common type of conjugate formation is coupling

with glucuronate:

UDP-glucuronate Phenol Phenylglucoronic acid

The other type of conjugate formation is the biosynthesis of sulfate esters with the
help of phosphoadenosine phospho sulfate (PAPS), the -active sulfate:

OSChH
Adenosine — Ribose — ©— O—SO3H

PAPS indoxyl indoxyisuifuric acid

Saks of indoxylsulfuric acid arc excreted with urine in trace amounts and
referred to as “animal indicant". Increased animal indicant in the urine reflects the
intensification of putrefaction in the large intestine.
Amide formation with glycine and glutamine plays a considerable role in
conjugation. For example, benzoic acid conjugation with glycine forms the more
soluble and less toxic hippuric acid (.V-bcnzoylglycinc), the measurement of which
excretion is used to determine the liver function:

COO- CO—HN —CH2— COO"

Benzoic acid Hippuric acid

562 Chapter 10. Tissue metabolism. Liver detoxification function

The conjugates arc eliminated from the liver either by the Bihary route — i.c.T by
receptor-mediated excretion into the bile or by the miai router via the blood and
kidney by filtration.

103. BIOTRANSFORMATION OF MEDICINES

The most medicines must be strictly dosed and their activity terminates in the
definite time after intake. The termination may be the re still of excretion from the
body in an. unchanged state (that is characteristic for hydrophilic compounds) or the
drug undergoes chemical modification {biotransformation) and as modified product
excreted from the body.
Biotransformation of medicinal agents includes:
1) the inactivation of medicines that is the decrease of their pharmacological activity
(phenobarbital, nitrites, ephedrine, etc.);
2) the increase of drug activity (butadion, mclhyldopa, normorphi.no. lovastatin,
etc.);
3) the appearance of metabolites that arc toxic for the human body (phcnacetin).
Drag inactivation usually occurs in two slops.
► First stage is the chemical modification under the action of monooxygenase
system of endoplasmic reticulum (microsomal oxidation), hydrolytic reactions
(Fig. 10.6).

Fi^ 10.6. Some cnemicai modifications: A — bar Disrate oxidation: B — aspirin hydrolyzes
10.4. MetaDolism and Detoxification of Ethanol 563

► The second stage is the conjugation of drugs (either native or modified by first
stage) with glycine, acetate, glycuronatc, sulfate, glutathione, etc. (Fig. 10.7);

Fig. 10.7. Conjugation reactions ol some drugs wim glucuronic acid: A — acetaminophen conjugation;
H — salicylic acid conjugation

Reduction reactions of some nitrocompounds (Fig. IO. 8):

C^ttorampharoco] AryJ amine metaioli’e

Fig. 10.8. Chloramphenicol detoxification

rno2-rnh2.
Some medicine doses require increasing because of their activity goes down in
permanent intake that occurs due to the induction of enzymes of monooxygenase
system and conjugation reactions. Exactly these circumstances lay in drug addiction
and some poisons. Broad substrate specificity of detoxification system enzymes has a
particular significance as well.

10.4. METABOLISM AND DETOXIFICATION OF ETHANOL

The endogenous alcohol is synthesized in the human body in glucose metabolism
(glucose pyruvate -»■ acetaldehyde -* ethanol) and as a rcsulL of cthyl-alcohol
564 Chapter 10. Tissue metabolism. Liver detoxification function

fermentation by intestinal and respiratory tract microbes. The source of exogenous

alcohol is hard liquorsand even food products (juices, bread, and kefir).
20-30% of ethanol is absorbed in stomach., 70-80% - by small intestine reaching
maximal concentration in blood in 30-60 minutes. Ethanol oxidation begins in mouth
mucosa and continues in many tissues, but 70-95% of ethanol is oxidized in the liver.
Ethanol is initially oxidized by cytosolic alcohol dehydrogenase (ADH) to form
ethanal (acetaldehyde). Further oxidization in mitochondria catalyzed by aldehyde
dehydrogenase, leads to an acetate formation (Fig. 1.0.9). With the help of acetate-CoA
ligase (synthetase) (ADLH), acetate is then converted to acetyl-CoA which is oxidized
in TCA to CO? and H,O. Abundant acetyl-CoA (in chronic alcoholism, for example)
is used for fatty acids, TAG and cholesterol synthesis in the liver. 10% of alcohol is
excreted unchanged with exhalant air, urine, and sweat.

IMADH +H NADH + H
H H
I I
I- 0 C O H
I I
H H
Etnanoi

Fig. 10.9. Ethanol oxidation

Genetic variations in the activity of alcohol dehydrogenase and aldehyde

dehydrogenase result in aldehyde accumulation that causes typical facial flushing,
chest pain, and hypotension. This is the underlying explanation for the alcohol
intolerance observed in Asian populations. Genetic polymorphisms co nt rolling alcohol
metabolism may predicate the extent of alcohol handled by the liver and consequently
the metabolites delivered to peripheral tissues that ultimately lead to injury.
Acetaldehyde is the first and most potent metabolite of ethanol, being highly toxic,
mutagenic, and carcinogenic. Acetaldehyde reacts with protein functional groups
(NH,S SH, OH), enzymes, receptors, glutathione, as well, as DNA (forms adducts)
damaging their function inducing oral cavity, throat, and urinary tract cancer.
Acetaldehyde inhibits NADH dehydrogenase decreasing ability of Hb to transport
oxygen and breaking energetic metabolism and ATP production. Acetaldehyde reacts
with dopamine and serotonin-producing opioids reacting with opiate receptors and
inducing alcoholic euphoria and inclination to alcohol use.
The microsomal ethanol oxidizing system (MEOS: particularly the enzyme
cytochrome P-450 2 E-1) is highly inducible by ethanol and promotes the conversion
of alcohol to acetaldehyde. However, this metabolic pathway generates free
radicals, reactive oxygen species, and oxidative stress, which arc responsible for
the hepatotoxicity and cardiotoxicity of alcohol. Some ethanol is also metabolized
by catalase. The role of microsomal ethanol oxidizing system (MEOS) in ethanol
metabolism is greater when large quantities of ethanol arc consumed (Fig. 10.10).
Consequences of chronic ethanol detoxification include: ketosis, hypoglycemia,
hypertriglyceridemia, hyperuricemia, insulin resistance, inhibition of mitochondrial
oxidative phosphorylation.
10.4. MetaDolism and Detoxification of Ethanol 565

H H NAD* NADH H
I 1
I 1
H—C--C— —H H—C
1 1 ADH 1
H H H

Fig. 10.10 Ethanol oxidation By MEOS: ADH — alcohol denyarogenase; MEOS — microsomal esnanoi
oxidizing system; ALDH — aldehyde dehydrogenase

Alcohol oxidation in the liver induces a high NADH/NAD' ratio in hepatocytes.

Lactate dehydrogenase reaction is shifted io lactate formation that results in increased
lactate content, and decreased pyruvate that causes reduced gluconeogenesis,
hypoglycemia and lactic acidosis.
The very high NAD H/NAD' ratio shifts all of the oxaloaccLatc in the TCA cycle to
malate, leaving the oxaloacctatc levels too low for citrate synthase to synthesize citrate.
The acetyl-Co A enters the pathway for ketone body synthesis instead of the TCA
cycle. Although ketone bodies arc being produced at a high, rate, their metabolism in
other tissues is restricted by the supply of acetate, which is the preferred fuel. Thus, the
blood concentration of ketone bodies may be much higher than found under normal
fasting conditions.
Ethanol-related high levels of NADH+H* and acctyi-CoA in the liver lead to
increased synthesis of neutral fats and cholesterol. However, since the export of these
in the form ofVLDLs is reduced due to alcohol intoxication, this leads to the storage
of lipids in the liver and fatty liver development (Fig. IO. 11).
566 Chapter 10. Tissue metabolism. Liver detoxification function

Fig. 1 QJt Toxic effects of alcohoLADH—alcohol dehydrogenase: ALDH—aldehyde dehydrogenase;

ROS — reactive oxygen species: MEOS — microsomal ethanol oxidizing system; DHAP —
dihydroxyaceione phosphate; LOH — Eactaie dehydrogenase; VLDL — very low density lipoproteins.
Alcohol oxidation produces toxic acetaldehyde (1}, wnich forms adducts wim amino acid, sulfhydryl
groups, nucleotides, and phospholipids causing hepatitis, indued on of MEOS (2) increases the
formation of free radicals (3). which leads to lipid peroxidation and causes irreversible liver cell
damage. The generation of NADh produced Dy alcohol and acetaldehyde oxidation greatly increases
the NADH/NAD*(in red) (4,5}.The elevated NADH/NAD* ratio conseguence is that me Dafance in me
lactate dehydrogenase reaction (6) is shifted toward lactate (7), resulting in lactic acidosis (3). The
increased NADH/MAD1 ratio cause hypoglycemia (9): pyruvate formed from alanine is converted to
lactate and cannot enter gluconeogenesis. The nigh naDh/naD^ ratio also prevents other major
gluconeogenic precursors, such as oxaloacetate and glycerol, from entering me gluconeogenic
pamway. The high NADH/NAD' ratio inhibits the oxidation of fatty acids and me TCA cycle, resulting
in the accumulation of fatty acids and favors if iacyl glycerol formation (10) (and development of
fatty liver). The triacylglycerols are incorporated into VLDL (very-low-density lipoproteins), which
accumulate in the liver and enter the blood, resulting in an emanot-induced hyperlipidemia (11)

10.5. CHEMICAL CHARCINOGENESIS

Oncogenes arc cellular genes that can trigger uncontrolled cell proliferation if their
sequence is altered or their expression incorrectly regulated.
10.5. Chemical Cnarcin agenesis 5&7

UncoH/roJW ceilpro/i/cratwn is an important indicator of tumor presence. While

normal cells in cell culture only divide 20-60 times, tumor cells are potentially
immortal and arc not subject to contact inhibition. It is known hundreds of genes
(proto-oncogenes} mutation of which, may promote the conversion of normal cell to
tumor one.
Almost every tumor begins with damage to the DNA of an individual cell The
genetic defect is almost always caused by environmental factors. These can. include
tumor-inducing chemicals (aitfflgras — c. g., components of tar from tobacco),
physical processes (c.g.. UV light. X-ray radiation), or in rare cases tumor viruses
(Table 10J)
Table 10J. Some chemical carcinogens

Polycyclic aromatic Exhaust gases, products at burning,

carbohydrates cigarette smoke, chemical-recovery
Benzanthracene, methylcholantrene,
industry
di methylbe nzantracen e
Aromatic amines Methylaminobenzene, naphtfiyfamine Color indu stry

Dioxins Tetrachloride benzodioxin Production of defoliants and growth

compounds, celiulose-paper
industry, water chlorination, burning
dumps

Mycotic toxins Aflatoxin B, Mold fungi

Nitrosamine Dimethylnitrosamine, diethylnitrosam ine Formed from nitrate-containing food

Carcinogens arc organic, as well as inorganic molecules: carcinogen icily is not

linked with a definite structural peculiarity. For example, polycyclic hydrocarbon
benzanthracene undergoes hydroxylation in the body, and its bypassed product is
carcinogen epoxide.
Covalent modification of guanine in DNA under the action of nitrosamines
(formation of N7-mcthylgLianinc or O6-mcihyIguaninc) leads to the rupture of
hydrogen bond between G—C in DNA chains and following alteration of DNA
primary structure causing damages of DNA interaction with proteins.
High doses of nitrates are identified as inorganic carcinogens. These compounds
arc widely spread in water and soil entering there from different sources, from fertilizer
agents, as well. They enter the human body with food (milk tinned foods, fruits,
vegetables) and as medicines. Nitrates and. in particular, nitrites are strong oxidants.
During the course of consecutive metabolic reactions nitrogen of nitrates binds eight
electrons. The intermediary products of these reactions can lake part in oxidation
reactions:
+5 +3 +2 +1 -1 -3
+2e +fe +le +2e +2e
hno3 —► HNCb NO —- HNO —* NH?0H —► nh3
Nitrates Nitrites Nitrogen Nitroxyl Hydroxylamine Ammonia
oxide

Oxidized substrates may be all iron-containing hemo prole ins: Hb, ETC
cytochromes, cytochromes of monooxygenase system of endoplasmic reticulum.
568 Chapter 10. Tissue metabolism. Liver detoxification function

Niirosammcs R.N-N=O formed in the body from nitrous acid (HNO,) and
second amines (R.NH) are sufficiently strong mutagens inducing alkylation (for
example methylation) of DNA nitrogenous bases. Nitrites convert cytosine residue
to uracil in DNA chain, as well, and the complementary pair GC becomes UC:

Cytosine uracil
During the course of replication of mutant DNA U forms complementary pair UA
which converted to AT in the next replication. If mutation occurs in proto-oncogcnc of
protein responsible for regulation of cell cycle, the next replication can lead to damage
of its structure, uncontrolled cell division and tumor development.
Nitrates and intermediary products of their conversions decrease the activity of
some enzymes of antioxidant system that leads to accumulation of reactive oxygen
species (ROS) and lipid peroxidation activation.
Aromatic amines. Aromatic amines arc compounds which are used in. the production
of aniline colors and resins. One of them is 2-naplnytamine which converted to
carcinogen 2-amino-l-naphtol in the liver. However, it conjugates with PAPS (or
Ul)P-glucuronic acid) and is converted to neutral compound in hepatocytes. By
excretion some conjugates arc hydrolyzed and that loads to formation of carcinogen
again which can induce the development of urinary bladder cancer (Fig. 10.12).

2-Naphthylamine absorbed by skin,

CO'" mouth or lungs

Oxidized in liver to 2-amino-1 -naphthol

Immediately conjugated with glucuronic

acid and excreted as glucuronide in urine

The glucuronide is hydrolysed in urine

by b-glucuronidase to liberate the
carcinogenic 2-amino-1 -naphthol

Fig. 10.12. Detoxification of naphtylamine with the following hy Orolyzis in urine

10.5. Chemical Cnarcin agenesis 569

Aflatoxins arc metabolites of some mold fungi, for example Aspergillus Jlavus
which arc developed in wrong storage of cereal products, groats and nuts Aflatoxin
I? I is converted to epoxide as a result of chemical modification in the liver which is
carcinogen and induces the development of liver cancer (Fig. 10.13).

Aflatoxin G 0.9-D ihydf O-8-(N' -gu3riy1)-9-nyCkOxy-

Aflatoxin S-Addukt

DNA/RNA
CYP450

Aflatoxin B-a.9-epoxide

Fig. 10.13. Aflatoxin B1 oxidation and Dinning to ONA (RNA|

Situational problems
1. Some amount of benzol during technical processing in a shoe factory was
evaporated. The workers inspired it with the air. Wliat mechanisms are employed
by the liver to gel rid of toxic compounds? To answer the question:
a) point, out the phases of toxic compound detoxification:
b) write down rcactionsofhcnzofdctoxilication, specify enzymesand coenzymes.
2. Epidemiological evidence suggests that diets high in meat and fat and low in
fermentable carbohydrate increase the colorectal cancer risk. One mechanism
that could explain the association with meal is increased colonic protein
metabolism due to increased protein, intake from high meat diets. Products
of colonic protein degradation and metabolism include ammonia, phenols,
indoles and amines which have been shown to exert toxic effects in. vitro and in
animal models. Why metabolites from colonic protein metabolism contribute to
this increase in genotoxicity during high meat intakes? To answer the question:
al write down the formulae of amino acids these toxic compound arc formed
from, and reactions of these toxic compounds formation;
570 Chapter 10. Tissue metabolism. Liver detoxification function

b) write down in formulae the detoxification reactions of these compounds.

c) present die scheme of aspirin inactivation.
3. The Czar Mithridates (Crimean realm} systematically took plant poisons in
small quantities to avoid acute poisoning. What is «Mithridates effect* based
on? To answer the question:
a> name stages of detoxification of compounds in the liver;
b) write down the scheme of detoxification of toxic compounds (in general
form), specify enzymes and coenzymes;
c) explain, why the systematic intake of small doses of plant poisons permits to
avoid acute poisoning.
4. Chloropicrin (C13CNO2) is a liquid with particular pungent odor and it toxic
for humans in large amounts causing lachrymatory and strangulating effects.
In the human body, chloropicrin is detoxified by glutathione transferase and
glutathione. Chloropicrin was used in First World War as a chemical agent
weapon. But in multiple uses, the damaging effect was sharply decreased on
humans and chloropicrin employment stopped in hostilities. Why the body
became resistant to chloropicrin? To answer the question:
a) specify the stages of toxic compound detoxification;
b) explain the mechanism of detoxification with glutathione transferase and
glutathione;
c) write down the reaction of chloropicrin detoxification:
d) give other compounds detoxifying with glutathione transferase.
5. Health effects associated with alcohol intake in large amounts include an
increased risk of alcoholism, malnutrition, chronic pancreatitis, alcoholic liver
disease, and cancer. In addition, damage to the central nervous system and
peripheral nervous system can occur from chronic alcohol abuse. Even light
and moderate alcohol consumption increases the risk of certain types of cancer.
What products of ethanol metabolism induce metabolic, toxic, narcotic and
carcinogenic effects leading to an internal organ and nervous system damage?
To answer the question:
a) write down reactions of ethanol metabolism in the human body:
b) specify two systems involved in ethanol metabolism;
c) explain the reasons for alcohol toxic effects.
6. It is well known the fact that decreased effectiveness of medicines activity, as well
as narcotic dmgs in surgery, is observed in prolonged drinking persons. Why is
the change in the rate of medicinal agents1 biotransformaiion observed in these
persons? To answer the question:
a) write down ethanol metabolism in the liver;
b) explain the ethanol influence on. microsomal oxidation activity in the liver.
7. Polluted town air, coaltar pitch, and tobacco smoke include polycyclic
hydrocarbons triggering oncological diseases. What arc the waste metabolites
formed in the body from polycyclic hydrocarbons with microsomal system
participation?
a) write down the scheme of polycyclic hydrocarbons catabolism:
b) specify cell component this product influence on;
c) explain the mechanism for its carcinogenic action.
10.5. Chemical Cnarcin agenesis 571

8. Water-nitratc (well-water) methemoglobinemia is the disorder developed in the

usage of water with large quantities of nitrates. I t is possible fatal outcome in
suckling child accompanied by expressed tissue hypoxia (cyanosis of lips and
cutaneous covering, shortness of breath). What biochemical processes damage
underlies in the base of methemoglobinemia development? To answer the
question:
a) write down the scheme of nitrate metabolism in the human, body;
b) specify, which compound structure nitrate metabolism products influence on
in baby's tissue;
c) explain the cause of hypoxia observed in. kids.
9. Aflatoxin I? I inducing primary liver cancer is the product of vital activity of mold
fungi Aspe^iffus which develops in the wrong storage of cereals and nuts.
What is the biochemical mechanism of the canccrogenic effect of aflatoxin 111?
10. Anti-fever and painkiller phcnacctin was administrated for many years. Its
metabolism occurs as follows:

Paracetamol or Panadol
The main ptarmacologkaB^
active metaMite

Hut phcnacctin production was discontinued due to its side effects and
oncological disorders induced in prolonged usage. Paracetamol is less toxic and
rarely causes complications in. therapeutic doses; it’s faster metabolized in the
fiver and excreted with, urine. Why is paracetamol less toxic and faster excreted
from the body? To answer the question:
a) examine these medicine formulae and explain the less toxicity of paracetamol;
b) write down reactions of paracetamol detoxification:
c) specify the oxidative product of hemoglobin formed in prolonged treatment
by phcnacctin or in poisoning by large doses of paracetamol.
11. Isoniazidum is widely used in t u be re u los i s prolonged therapy. Why this medic inc
induces side effects and complications (for example, neuritis, damages of optic
nerve)? To answer the question pay attention to reactions linked with this
medicine:
572 Chapter 10. Tissue metabolism. Liver detoxification function

isoruazktum inhibits the enzyme catalyzing the reaction:

a) specify compound formulae of which arc present in these reactions; point out
biological importance of these compounds;
b) specify enzyme catalyzing the second reaction and its class;
c) point out the biochemical derangements which, can occur in patient body as a
result of reactions with isoniazid:
d) name vitamin, which, have to be administrated in prolonged treatment by
isoniazid.
 INDEX

A .Alanine (Ala) 12, 141, 144,401

Abctalipoprotcincmia 318 Alanine aminotransferase (ALT) 101, 104,
ABO blood groups 365 228, 275
ACAT (acvl-CoA — cholesterol acyl trans­ Albinism 103, 176,440
ferase) 371, 373 Alcohol dehydrogenase 79. 100. 302, 564
Absorption 76. 154, 253 Aldehyde dehydrogenase 302. 564-566
Absorptive state (Fed state) 259, 278, Aldosterone, action 539, 540—542
509-511 Alkaptonuria 440
Acetate 302, 303, 557, 564 Allopurinol 104, 476
Acetaldehyde 100. 302, 564, 566 Allosteric center (site) 43, 61, 94
Acetaldehyde dehydrogenase 303, 303 Allosteric regulation 94. 422
Acctoacctatc 345—348. 433. 528 Alternative splicing 135, 170
Acetyl-CoA carboxylase 324, 325, 327, 341 Alzheimer’s disease 52, 54
Acetylcholine 30, 98, 104— 106, 198 a-Amanitin 154
Acetyl-CoA 220-224, 266 Amino acid(s) (See also specific types,
Aconitase 223—225, 243 e-fi-)
Active site (active center) 26, 27, 43, 57 Alanine 156. 187, 394
Active sulfate 558, 561 catabolism 78, 220, 433, 507
Active transport (See Transport) 194-196 classification 10, 12
Acyl-CoA 315, 330, 340-343 glucogenic 276, 433
Acyl-CoA dehydrogenase 341, 343, 350 non-essential 395, 404, 441
Acyl-CoA synthetase 315, 340 Aminoacyl-tRNA 142, 143-145
Adenine nucleotide translocase 241 Aminoacyl-iRNA synthetase 143, 144, 148
Adenine phosphoribosvl transferase 106, y-Aminobutyratc (GABA) 424, 450
472 Aminopeptidase 397, 401
Adenosine HO. 446 Aminotransferases (transaminases) 60,
Adenosine 3’-phosphate-5’-phosphosul- 101,404, 434
fate ( PAPS) 558, 561,568 Ammonia detoxification 413, 415
Adenosine deaminase, deficiency 476 Ammonia salts 413, 415,
Adenosine triphosphate (ATP) 92, 216. AM P — activated protein kinase 200, 328,
217,371 375
ATP synthesis 220, 226, 230 Amphibolic pathway(s) 228, 229
ATP synthase 230, 231, 239 Amylase 60, 78, 255
ATP-ADP cycle 216,217 Anabolic pathway(s) 218, 228
Adenylyl cyclase 198-202, 214 Ana plerotic reaction(s) 228, 409
Adipose tissue (Sec also FAT, TAG) 244, Andersen's disease 301
245, 252 Androgens 487, 489, 493, 503-505
ADP/ATP translocase 241. 243 Angiotensin 16, 540—543
Adrenocorticotropic hormone (ACTH, Annexin-1 (or lipoconin) 361
corticotropin) 485, 493, 496 Anticodon 115, 116, 143
Affinity chromatography 49, 51, 52 Amidiurctic hormone (ADH) 16, 485.
Aflatoxin B 569 486, 535
ALA (Aminolevulinic acid) 460 Anti oxi danl(s) 15, 73,, 285
ALA dehydratase 459, 461 Antiport 195, 196
ALA synthase 459—461 Apoenzyme 60
574 index

Apoprotein(s) 316—318 Calmodulin 26, 203. 204

ALAI! 384 cAMP (cyclic 3r,51adenosinc monophos­
B-100 331, 387 phate) 207, 213
B-48 316, 320 Cap structure 132, 146
CII 381,384-386 Capping 132
.Apoptosis 121, 127, 151 Carbamoyl phosphate 418, 422, 469
.Arachidonic acid 311, 313. 354 Carbamoyl phosphate synthetase I 418,
Arginase 419, 422 422, 469
Arginine 23,422 Carbamovl phosphate synthetase 11 469.
Argininosuccinasc 4! 9. 425 471
Argininosuccinatc 419. 425 Carbohydra Lc(s) 29, 253, 365
Argininosuccinatc synthetase 419, 422 Carboxypeptidases A and B 399, 401
.Aspirin (acetylsalicylic acid) 100, 562 procarboxypeptidases A and B 399
Asparagine 152, 228, 431 Carcinogens 124, 558, 567
Asparagine synthase 228 Cardiolipin 364
.Aspartate 46, 228, 416 Carnitine 15, 220, 340
Aspartate aminotransferase ( AST) 102. Carnitine palmitoyl transferase I (CPT I)
404 343
Aspartate transcarbamoylase 471 Catabolic pathway! s) 221, 252. 404
Atherosclerotic cardiovascular disease 372 Catalase 246, 248
ATP (See Adenosine triphosphate) 216, Catalytic receptors 205, 207. 209
217 Catalytic site 60, 205, 206
Atrial natriuretic factor 542 Catalytic specificity 64
Catccholaminc(s) 436, 508
B Cathepsins 403
Beriberi disease 75, 243 Cellular respiration 229, 243
BH4(Scc Tctrahydrobioptcrin) 436, 439, Cellulose 84, 254
440 Ceramide 191, 312, 365
Bi functional enzyme 280, 288 CETP (Cholesterol ester transfer protein)
Bile salts. Conjugation 313, 319, 378 385
Bilirubin 463, 464 eGM P (cyclic guanosine monophosphate)
conjugated (digtucuronidc), direct 464 209
unconjugated (indirect, nonconju­ Chenocholic acid 377
gated) 465 Cholccaicifcrol (vitamin Dp 73, 370
Biliverdin, 462 Cholecystokinin 379, 399
reductase 462 Cholesterol
Binding site 60, 486, 487 synthesis 104, 383. 387
2;3-Bisphosphog]yceratc (2,3-BPG) 42 regulation of 370, 393
Body mass index (BM I) 333 reverse transport 385
Bohr effect 40 transport 385
Branching enzyme 290, 299 Cholesterol esters 313, 316, 331
Brown adipose tissue ( BAT) 244, 252, 503 Cholic acid 377
Choline 73, 445, 452
C Chorionic gonadotropin (hCG) 495
CAD enzyme 471 Chromatin 165, 180
Cadaverine 560 Chylomicron(s)T (CM) 316, 331, 381
Caffeine 201, 306, 352 mature. (mCM) 381
Calcitonin 489, 547 nascent (nCM) 381
Calcitriol 213, 546 Chymotrypsin, 400
synthesis 546 Chymotrypsinogen 399
index 575

Citrate 223, 228 Cytochrome P450 (CYP450) 344, 377,

Citrate synthase 223, 228, 244 555
Citric Acid Cycle (CAC) Sec also Tricar- Cytochrome P450-dc pen dent monooxy­
bocylic Acid Cycle (TCA cycle) 220. genase enzyme(s) 555
223, 228
Citrulline 418, 425 0
Citrull incmia 425 DAG (diacylglyccml) 204, 205, 315
Co ASH 221, 340 Deamination
Coated pits 193. 195, 383 amino acids 403, 406
Coenzyme Q 231, 232, 341 indirect 407, 408
Cofactor 60, 72 non-oxidative 407
Colipasc 313, 319 oxidative 406, 407, 421
Combined immunodeficiency (SCID) Debranching enzyme 291, 292, 30!
476 Decarboxylation of amino acids 450
Common Catabolic Pathway (CC P) 221., Dehydrogenase(s) 79. 221, 228
252 Deletion 18, 172
Compartmentalization 92 Dextrins 254
Complementarity 62, 200 Diabetes insipidus 539
Complex enzymes (holoenzymes) 60, 86. Diabetes mcllitus 53, 347, 526
87 acute complications 529
Co proporphyrinogen 111 460 late complications 531, 532
Co-repressors 159 non-insulin dependent (N1DDM) 526
Corticosteroid(s) 504, 506, 524 Dihydrofolatc reductase 80r 100, 471
Corticotropin (ACTH) 491, 496 Dihydrobiopterinc reductase 439
Corti co tropin-releasing hormone (CRH) Dihydroorotasc 471
491, 496, 526 Dihydroorotatc dehydrogenase 471
Cortisol. 422, 433, 504-508 Dihydroxyaccionc phosphate (DAP) 274,
secretion 507. 519, 525 328
synthesis 504 2,4-dinitrophenol (DNP) 245
Cory cycle 276, 522 Dipeptidase 410
Cory disease 301 Diphtheria toxin 154
C-pcptidc 498, 518 Direct Reversal Repair 124
CRE (cAMP response element) 201, 202 DMA (deoxyribonucleic acid) 108. 127-
Creatine 15, 442. 447, 448, 522 132
Creatine phosphate 447. 522 DNA dependent RNA polymerase 129
Creatine phosphokinase 102 DMA double-stranded breaks 1.27
CREB (cAM P response element binding DNA ligase 1.21, 127, 184
protein) 167, 201 DNA polymerase 119. 122. 183
Cyanide 242, 243 DNA replication 100, 117-123
Cyclooxygcnasc(s) 1.00, 357, 360 DOPA decarboxylase 436, 441
' COX-1 357, 360
DOPA oxidase 437
COX-2 357, 360 Dopamine 54, 436, 440, 441
CYP2EI 556 Dopamine hydroxylase 436
CYP3A4 555, 556
Cystic fibrosis 135, 172. 179 E
Cytochrome b-cl (Complex 111) 237, 238. Eicosanoids 308, 311, 313, 354
242 Eicosapcntanoic acid 311. 356. 360
Cytochrome c 174, 234, 242-244 Eicosalricnoic acid 355, 356
Cytochrome c oxidase (Complex IV) 234. Elastase, 397, 399, 401
' 238,244
Proclaslasc 399
576 index

Electron transport chain (ETC) 174. 229,, Feedback inhibition 95, 328, 374
421 Ferrochelatase (heme synLhasc) 460. 461
Electrophoresis SO. 182, 264 Fibrous 29
Embryonic hemoglobin (Sec Fetal hemo­ Flavonoid(s) 248
globin) .35, 36 5-lluo ro uraci I. (5 - F U ) 478
Endergonic reaction 218 FMN 60,75
Endopeptidase 397 Foam cell(s) 387-389, 393
Enhancer 167, 210 Folate reductase 443. 444
Enoyl. Co A hydratase 341 Folic acid (vitamin Be or vitamin Bg) 99,
Entero hepatic circulation 154. 379. 389 443-445
Entero hepatic urobilinogen cycle 464 Follicle-stimulating hormone < FSH > 485,
Enteropeptidase 399 492, 493
Enzyme 57 Formyl-H4-folate 444. 468
activity 65—67, 92 Fructose 96,. 279-281
classification 78 Fructose 6-phosphate 266, 274
clinical applications 100 Fructose -1.6-bisphosphatc phosphatase
induced fit model 65 279,281
lock-and-key model 64, 65 Fructose-1.6-bisphosphatc 279, 281
regulation 92, 93 Fructose-2,6-bisphosphatc 96, 279
specificity, stereo 64 Fructosc-6-bisphosphatasc 274
V (maximal velocity) 66, 67 Fructose-6-phosphaie 266, 274, 279
unit of activity (U J 69 Fumarase 227, 243
Enzymopathies 103 Fumarate 97, 227. 433
Epinephrine (adrenaline) 1.5, 197, 201. 295
Ethanol 100, 302, 563-566 G
Ethanolaminc 309, 329 Galactocc re broside 313
Ethanolaminc mcthyllransfcrasc 452 Gallstones disease (cholclilhias) 379, 400
Euchromatin 165 Gangliosidc(s) 191, 313, 367, 369
Eumelanin 437 Gastrin 16, 398,492
Exergonic 218 Gaucher’s disease 369
Exogenous cholesterol 381 Gel electrophoresis 182, 184
Exon(s) 135, 170 Gel filtration chromatography 51
Exopeptidases 397, 400 Gene cloning 179, 184
Gene rearrangement 165, 166
F Gene regulation 159, 166, 170
FAD (Flavin adenine dinucleotidc) 60, 75, Genetic code 141 — 144, 156
555 Gierke’s disease 299
Familial lipoprotein lipase deficiency, dis­ Glycogen 93, 254
lipoproteinemia type I 319 synthesis 289, 290
Farber’s disease 369 degradation 293, 289
Fasting state, (Poslabsorptivc state) 259, hormonal regulation 353, 509
374 Glucagon 280, 294, 500
Fat (See also TAG) 313-319 action 343
Fat malabsorption 319 biosynthesis 500
Fat-soluble vitamin(s) 73, 308. 313 Glucoamylase complex 255
Fat tv acid (s) 308-312, 315 Glucoccrcbroside 333
essential 311, 319,327 Glucocorticoids 361, 504. 508
structure 325 Glucokinase 259, 500, 511
synthesis 323, 328, 500 Gluconeogenesis 78. 271-282
index 577

Glucose 104, 159 G ua n i n c 110, 472—475

Glucose I-phosphate 86, 290 Guanylyl cyclase 209, 542
Glucose 6-phosphate dehydrogenase 282.
285, 289
H
Glucosc-6-phosphatasc 274. 299 Haemophilia A 179
Glucosc-6-phosphate 266, 278, 300, 511. HbA (hemoglobin A) 35, 43. 44
Glucosc-al.ani.nc cycle 276. 415. 417 HbC 178
G lucose-l ac late cycle 276 HbF 18, 43.44
Glucuronyl transferase 464 HbS 44-46, 178
GLl;T=glucose transporters 256 Hb Ale 35
Glutamate (Glutamic acid) 44, 407, 431 Hb A2 35
Glutaminase 414. 415. 418 Heinz bodies 285, 288
G lutamine synthetase 414, 423 Helicase 119, 120
y-Ghitamyl transpeptidase 402. 413 Heme 34-37, 171
G1 utat hione (glutam vl-cvstcinvlglvci ne) oxygenase 461
15, 285,402, 558' synthesis 459, 460
Glutathione peroxidase 248, 559 catabolism. 461
G lutathione S-transferases 558 Hepatic triacylglycerol lipase (HTGL).
Gluten intolerance 396, 397 318, 386
sensitivity 396 Hereditary diseases44, 128. 176, 179
Glyceraldehyde-3-phosphaLc 263, 265 Hereditary hemoglobinopathy 44
Glycerol. 3-phosphaLc 328-330. 500 Hereditary orotic aciduria 471
Glycerol kinase 274, 328 Hers’s disease 301
Glycerophosphate shuttle 263 Heterochromatin 165
G lycc ro phospholi pids (phosphoacylglyc - H exo kinase 259, 266, 290
crols) 312, 362-365 Hippuric acid 425, 561
Glycine 1.5, 379, 401 Histamine 451,452
cleavage enzyme 442 Histidine decarboxylase 79
synthase 442 Histidinemia 407
Glycochenocholic 379 Histones 151. 162, 165
Glycocholic acid 379 HMG CoA (p-hvdroxv-p-mcthvlglutary!
Glycogen 93, 254T 289 CoA) 345-347, 372, 374
Glycogen phosphorylase 291.292, 298 HMG CoA.lyase 345
Glycolipid(s) 190, 191, 313, 365 H MG-CoA reductase 1.04, 374
Glycolysis, 219, 260 H MG-CoA synthase 345-347
aerobic 260, 265, 522 Holoprotcin 28
anaerobic 260, 522T 531 Homocysteine 447
Glycosidase 255 HomocysLinuria 447
Glycosuria 525, 527, 528 Homogentisate dioxygenase 437. 440
Glycosykransfcrasc 367 Homogen tisic acid 437, 440
G onadolropi n-rc least ng hormo ne Hormone sensitive lipase (HSL) 339, 528
(GnRH)485, 491 Human chorionic gonadotropin (hCG)
Gout 104, 475 485
G RAC IL E syndrorne 244 Hydrogen peroxide 82, 245, 246
Growth hormone (Somatotropin) 179, Hydrolases 78, 83
492-494 p - H ydroxybu tyrate 345—347
hypersecretion 523 Hydroxyl, radical (HO* ) 245-247
Growth hormone-releasing hormone p-Hydroxy acyl CoA dehydrogenase 341
(GHRH) 491, 492 7a-Hydroxylase 377, 378
578 index

21-Hydroxylase 504, 526 Inlron(s) 135-137

17-HydroKysteroid(s) 506 Inversion 172, 173
Hyperaldosteromsm 525, 542 Ion exchange chromatography 48, 49
Hyperammonemia Type I. Type 2 244. IP3 (inositol 1,4,5-trisphosphate) 195,
413, 423, 42S 204, 205
Hype rargin inc mia 425 Isocitratc 223, 228, 422
Hyperbilirubinemia 465, 466 Isoelectric point (pl) 46-48, 101
Hyperbilirubinemic toxic encephalopathy Isocnzynic(s) 227, 259, 317
' 467 Isomerases 79. 86
Hypercalcemia 547. 548
J
Hypercholesterolemia 73, 172, 372, 386
familial (genetic) 172, 390 JAK2 proteins 494
H yperc hylonticroitem i a 319 Jaundice 459, 465, 466, 481
Hypercortisol ism 524. 525 hemolytic (prehepatic) 466
Hyperglycemia 525,. 527, 531 hepatic 466
Hyperlipidemia 528. 529 mechanical (obstructive, post-hepatic.)
Hyperparathyroidism 548 466
Hyperthyroidism 448, 495, 524 neonatal * physiological jaundice* 466
Hypertriacylglycerolemia 299, 300,. 319
K
Hyperuricemia 475 , 482, 56 4
Hypocalcemia 430. 547, 548 Ketoacidosis 48. 345. 531, 535
Hypoglycemia 78 , 299- 303 17-kelosleroids 506, 517,
Hypoparathyroidism 548 Ketone bodies 345-349, 565
Hypothyroidism 377, 524 Kctonemia 345, 347, 528
Hypoxanthine 472—476 Ke ton u ria 347, 528
H ypoxanlh i nc-gu an ine p hosp hori bosyl- Km (Michaelis constant) 66, 67, 259
transferase 472—474, 476 Kozak consensus sequence 146
Krebs cycle (Sec TCA cycle) 76, 223. 422
I Krebs-Hcnsclcit cycle 418
Icterus 466 Kwashiorkor 338, 396, 410
IMP (inosinc-51-monophosphate) 468,
L
472-474
IMP-AMP cycle 407, 408 Lactate 260. 264-266, 276-278
Indirect deamination 407 Lactate dehydrogenase (LDH ) 33, 80, 264
Inhibition of Enzymes 90, 96, 100 Lactic acidosis 244, 266, 278, 531
competitive 97, 476 Lactose 159—161, 253, 260
irreversible 98, 100.360 Lactose intolerance 260
non-competitive 97 Lagging strand 120, 140
reversible 96 LCAT (lecithin cholesterol acyl
Insertion 124, 172, 180 transferase) 385, 386
Insulin, 205. 258 L-Dihydroorotatc 471
biosynthesis 497 LDL receptors 382-384, 386-388
receptor 197, 205, 435 L-DO PA 88, 440, 441
receptor substrates (IRS) 205 Leigh syndrome 244
signal transduction 213, 282 Leptin 16, 245, 332
Insulin-dependent diabetes mcllitus Lcsch-Nyhan syndrome 472. 473
(IDDMJS26 Lcucotricncs (LT>354. 355
Insulin-like growth factor(s) 487, 493, 494 L-Glutamatc dehydrogenase 406
InterferoB(s) 155, 355, 357 Ligand 26-30, 51
Intrinsic factor 398 Ligases 87. 165, 324
index 579

Lipase 78: 313. 317-319 Microsomal triglyceride transfer protein

pancreatic 63, 313, 319 (MTP)319,331
hormone sensitive 339, 528 Mineralocorticoids 504, 508
lipoprotein, lipase (LPL) 317—319, 381 Mismatch repair 121—123
Lipid peroxidation 246. 247. 365. 568 Missen sc mutation 171., 178, 187
Lipids !90, 253,208 Mitomycin 153
emulsification 313 Mixed micelles 315, 371, 378, 379
digestion 314, 315, 319 Monoacyiglyccrol (MAG) 314. 315
Lipoic acid 80,221-223 Monoamine oxidase < MAO) 1.04, 451,
Lipoprotein(s) 317—319, 38 ! 453
p-Lipotropin (P-LPH) 493, 496 Monooxygenase (hydroxylase) 104, 374.
Lipoxygenase pathway 357 563, 567
Lost coefficient 395 P-MSH (melanocyte-stimulating hor­
Luteinizing hormone (LH) 492.. 493 mone) 496
Lyases 86 mRNA. 115-117, 133, 135
Lysophospholipid! 315 mRNA (pre-mRNA) 132, 133, 135, 136
Lysosomal storage diseases 368 Mutation. 46, 151. 157, 171
nonsense 172
M missense 171-173
Mac Ardleys disease 301, 302 silent 171
Malate 64. 227, 565 I'ramcshifi 172
Malate aspartate shuttle 220? 227, 263 Myoglobin (Mb) 28, 33-36, 39
Malate dehydrogenase 64, 227. 272
Malondialdehyde 247 N
Malonyl CoA 324-327, 343 IS -Acctylglutamatc 422—424
MAP-kinase 207, 208 NAD1 /NA D H I n icotinam i d c ad c n i n c
Maplesyrupurincdisea.sc 179 dinuclcotidc) 228, 231
Marasmus 396 NADH dehydrogenase 235, 564
Medium chain fatty acyl CoA NADP /NAD PH 282
dehydrogenase (MCAD) 343, 344 N-Carbamoylaspartate 471
MEK-kinasc < mitogen-activated protein Negative balances 395
kinase kinase) 207, 208 Neuropeptide Y 332
Melanocyte stimulating, hormone 485, Niemann-Pick disease 369
496, 525 Nitric oxide (NO) 209; 243, 246
Melatonin 15, 445, 451 Nitrogen balance 395, 410. 411
MEOS (microsomal ethanol oxidizing Nitrosamine(s) 567, 568
system) 556, 564-566 Nonsteroidal anti — inflammatory drugs
Messenger (N SAI Ds) 100, 360
primary. 197, 200, 207, 209 Norepinephrine (noradrenaline) 436, 437,
secondary 199, 209, 214 458, 503, 508
Methemoglobin 46 Northern blotting 185
Meihcnyl-H^-folate 444, 468 Nuclease 85, 170
Methotrexate 100T 478, 482 Nucleic acid 108, 110, 180
Methylation 115, 151, 166 Sec DNA. RN A synthesis 110,471
McLhylcobalamin 82, 447 Nucleoside diphosphate kinase (NDP ki­
McLhylcnc-H4-folate 442. 444 nase) 469: 470
Methyl-H+-folate 444, 446. Nucleoside monophosphate kinase
M ct hy(transferase 133,446 (NMP kinase) 469-471
Mevalonate 372, 374 Nucleosome, structure 162, 163, 165
580 index

Nucleotide Excision Repair 122—124 Pentose phosphate isomerase 284

Nutritious (alimentary) value 395 Pentose phosphate pathway (PPP) 282
Pepsin 398-400
0 Peptide backbone 12, 18-20
Obese gene 333 Peroxidase 82.. 152, 357
Obesity 323, 332-334 Peroxisome 333, 344, 364
Ocular albinism 176, 177 Phenylalanine hydroxylase 435, 438
Okazaki fragments 120, 121 Phe nyl pyru vate 438, 439, 456
Oligomycin 230, 243, 248 Phosphatase 84, 92, 470
Oligopcptidc(s) 12, 409, 410 Phosphatidyl choline (lecithin) 363, 385,
Operator 158—161 448
Opcron 158-161, 188 Phosphatidyl cthanolaminc 363, 449
Origin 118, 119, 121 Phosphatidylinositol (PI) 364
Orlistat 323 Phosphal idyl inositol 4,5-bisphosphaic
Ornithine 418-422, 425, 433, 529 (P1P2) 202-204, 214
Ornithine cycle 418, 421, 425: 433 Phosphatidylcthanolaminc 312, 448
Ornithine transcarbamoylase 41.8, 425 Phosphodiesterase 200, 201, 206. 207, 298
Oro talc phosporibosiltransfera.se 470, 471 Phosphocnolpyruvate (PEP), 220, 272,
Orotidinc monophosphate. (OMPJ47I 274, 277
Orotidylic acid decarboxylase 470 Carboxykinase 274, 277
Osteomalacia 547. 548 Phosphofructo kinase 262, 266. 279-282
Oxaloacclatc 91, 223, 228. 272-276, 404, Phosphofructo k i nase - 2/fruc lose
565 2,6-bisphosphatasc (Bifunclional en­
Oxidation of fatly acids zyme) 279
a-oxidation 344 Phosphoglucomulasc 86, 290
p-oxidation 91. 92, 220 6-Phosphogluconaie 282. 283
cu-oxidation 344 6-Phosphogluconolactone 282
Oxidative decarboxylation of pyruvate 221, Phospholipase A, 315, 356, 361, 364, 365
251,252 Phospholipase C (PLC) 202, 204, 205,
Oxidative phosphorylation, 229. 230 365
coefficient 240 Phospholipid(s) 190-192, 312, 315-318
uncouples of 244. 245 Phosphop rotein phosphatase 207
Oxi doted uc tascs 79, 80, 82 5-Phosphoribosyl-1 - amine 468
Oxphos diseases 244 5-Phosphoribosvl-1 -pyrophosphate
Oxygen dissociation curve 39, 40 (PRPP) 467, 468
Oxvgcnasc(s) 80, 461, 462 Phosphorylation 92, 106. 151, 152, 169—
Oxytocin 16, 485, 490. 493, 497 171, 229, 259
PhosphoryJa tion/D c phos phorylation. 92..
P 106, 278, 327, 374
Palmitic acid 311, 324, 325, 363 P hotosc nsi ti vi ty 461
Pancreatitis 53, 100, 104,400 Phytostcrolcmia 371
Pancreatic trypsin inhibitor (See Trypsin) PKU (phenylketonuria) 438
399 Platelet-activating factor (PAE) 364
Para-aminobenzoic acid (PAHA) 443 Poly(A) tail. 130, 133, 146, 170
Parathyroid hormone (PTH) 485 Polyadcnylalion sites 170
Parkinson’s disease 54, 104, 441. Polydipsia 529-531
Pyruvate dehydrogenase complex (PDIIj Polymerase chain reaction (PCR) 180. 1.83
221, 225,251 Polypeptides 12, ISO, 186, 237,471.
PDH kinase 223 Polysome 150
phosphatase 223 Polyuria 529—531
index 581

POMC (proopiomelanocortin) 495 Pyridoxal phosphate (PEP) 403, 404

Porphobilinogen (PBG) 459 Pyrrole ring 34, 37, 459, 462
Porphyria, intermittent 460, 461 Pyruvate
Porphyrin 459, 460—462 ' carboxylase 78T 272, 277, 434
Postah sorptive state (fasting) 2SO, 293. kinase 262, 265, 281, 466
294, 511, 512
Post translational modifications 151, 152, Q
171, 193 Q cycle 237, 238, 242
PPARs — peroxisome proliterator- acti­
R
vated receptors 333
pre-mRNA 132, 133, 135 Reactive oxygen species (ROS) 245, 285,
Primase 119, 120, 139, 140 363, 568
Primer 119-121, 183 Recombinant DNA 179—181
Proinsulin (See Insulin) 497, 49S Reductase 80, 91, 374-377
Prolactin (PRL, lurcotropic hormone or Regulatory gene 160, 161
luteotropin) 485, 493 Renin 540—543
Prolactin-releasing factor 491 Renin-angiote nsi n -a Idostc rone system
Prolactostatin 491 (MAAS) 540
Promoter 130, 131, 159-161.. 166, 167 Repair. DNA
Prostaglandins 354—358 base-excision repair (BER) 124
PG IX 357 Replication 117-124
PGEJ57 Replication fork 119—121
PGF/1357 Replicon 118, 121
PG H. 357 Respiratory chain (See Electron Transport
Chain.'ETC) 229
Prostacyclin (PG I) 356-359
Prosthetic group 28, 33, 60, 72 Restriction
Proteases 85. 403, 500, 501 Enzyme 179-182, 1.84, 186
Protcin(s) Restriction fragment-length polymor­
classification 28 phisms 184
denaturation 24, 67 Reverse cholesterol, transport 317. 384-386
digestion 394-397. 399, 401 Riboflavin (vitamin Bz) 75, 234
domain 25, 26 Ribonuc I coprotein 28, 135, 1.36
inhibitor 29, 30 Ribonucleotide reductase 471, 472
glycation 387 Ribose-5-phosphate 282, 469
a-helix 19, 20 Ribosome 116, 117, 143-150
P-shcct 20-22 Ribulose 5-phosphate 282—284
levels of structures (primary, second­ Rickets 548, 551, 552
ary, tertiary, quaternary) 17 Ritz coefficient 405
Protein kinase 94. 199—205, 207. 209. 329 RNA
Protcinopalhy, primary 52, 53 Editing 1.70
Protein-protein interactions 94, 167, 168 Interference 170
Proteolysis, (limited, partial) 93, 397-399 pol II 130,133, 135
Protomer 28, 33, 35-39 polymerase 129, 130
Proton, electrochemical gradient 239, 241 polymerase 11 1.30, 131, 154
Protoporphyrin IX 34, 460 polymerase 111 154
PRPP synthetase 474 primer 119, 120. 121
Purine nucleoside phosphorylase defi­ splicing 135, 137
ciency 476 Rotenone 242. 252
Putrescine 560 rRNA 115-117
582 index

S I
S-adenosyl homocysteine (SAH) 446 TAG (triacylglycerols) 63, 84, 311, 31.3
S-adenosyl me ih ion inc (SAM) 435, digestion 315, 319
446-449 hormonal regulation 323
Salicylic acid LOO. 562,563 mobilization 519
Salvage pathway 467.^ 468, 472, 473 synthesis of 317, 330
Scatole 560 transport by lipoproteins 323
Scavenger re cepto r (S R) 384-387 TATA-box 130
Secondary bile sails, 379. 380, 392 Taurochenocholic acid 379
dcoxycholic 379 Taurocholic acid 380, 391
l ilac hoi i c 379 Tay-Sachs disease 369
Secondary proleinopathy 52, 53 TCA cycle (See also Krebs cicle) 78, 223,
Serine decarboxylase 452 227, 243
ScroLonin 15, 451, 453 Tctrahydrobiopterin (H+-hio pterin) 435
Sickle cell anemia 44. 45, 171. 178 Telrahydrofolate (THF, H^-folatc) 442
Somatostatin (SS) 485. 489, 491, 492 Thermogcnin 245. 252. 503
Southern blot 185, 186 Thiamine pyrophosphate (TPP) 74, 221.
Specific catabolic pathway(s) 218, 21.9 225
Specificity of transformations 64 Thromboxane (TXA) 354—360
Sphingolipids 191,308, 31.2, 362 Thymidylate synthase 471, 478
Sphingomyelin 191. 309, 312, 362 Thyroglobulin 501., 502
Sphingosine 77, 191, 312, 362, 365 Thyroid hormones (T1, T4) 169, 170, 194,
Squalene 373, 374 210, 493.501
SSB (single strand binding proteins) 119. receptors 169, 170, 487
120 peroxidase 524
Starch 53, 218, 253-256 Thyroid-stimulating hormone (TSH), or
Stearic acid 364 Thyrotropin 485, 493
Steatorrhea 319, 322 Thyrotropin releasing hormone (TRH)
Stercobilin 465, 466, 481 485, 491
Steroid hormones 169, 176, 282, 370, 382. Thyroxine (Tj 495, 501
503 Tophi gouty arthritis 475
Slop codon 141, 149, 172, 173 Topoisomerase 119. 153, 156
Substrate cycle 278-281 Transatdolasc 284
Substrate level phosphorylation 226, 265, Transaminase 79, 228, 403-405
522 Transcription 141, 153, 168
Subunit 33, 60, 94, 117, 145 Factors 26, 130, 131
Succinate dehydrogenase 226, 236 Transferases 79, 82
Succinyl CoA 220, 225, 226, 459 Transketolase 74, 79. 284
Succinyl CoA: accloacctale CoA Translation 113, 115, 133, 141-153
transferase 346 Translocation 147, 149, 173
Sucrase isomahasc complex 255 Transmembrane proton gradient 235. 245,
Sucrose 253, 255, 300 252
Sulfo lipid 366 Transmethylation reaction 445-447
Sulfonamide 99, 445. 458 Transport
Superoxide anion (O2 ) 245, 246 Passive 194
Superoxide dismutase (SOD2) 248 Active 194
Surfactant 312, 363, 364, 369 Triacylglycerol (TAG) 63, 311-319
Sympon 194-196, 501 Triiodothyronine (Ta) 501
index 583

lRNA(Scc RNA) 115-117 Vita mints)

Trypsin 397, 399 A. (Retinol) 73
trypsinogen 397, 399 Bj (Thiamine) 73
Tyrosine hydroxylase 436, 440, 441 B ( Riboflavin) 73
Tyrosine protein kinase (Tyr-PK) 205, B J.Niacin) 73
435 Bs (Pantothenicacid) 73
B. (Pyridoxine) 73
U
Bk (Inositol) 73
Ubiquinol 232, 236-238 B,3( BCr folic acid) 73
Ubiquinone 80, 232,238 B|# ( Para-aminobenzoic acid) 73
Ubiquiti nation 152, 153, 169 Bn (BT. carnitine) 73
Ubiquitous enzyme 469—471 B|3 (Cobalamin) 73
UDP-glucosc (uridine diphosphate C 73
glucose) 290, 291 D 73
U DP-glucuronic acid 464, 467, 557, 468 E73
11 brace n tri fuga t ion 319, 320, 321 F (Polyunsaturated fatty acids) 73
U M P (uridine monophosphate) 471 fat soluble 73
U M P synthase deficiency 471 H or B7 (Biotin) 73
Uncoupling protein-1 (UCP-l) 8, 246 K 73
Urea 63,413,418 P (Bioflavonoids) 73
Urea cycle 413, 418 waler soluble 73
Uric acid 106,300,413
Urobilinogen 464. 465, 466 W
Urobilin 463, 465. 466 We rnickc - Korsa kolTs sy nd tome 243
Uroporphyrinogen 111 459 Western blotting 185
Uroporphyrinogen 111 synthase 459
X
V Xanthine
Van den Bergh test 465 Xanthine oxidase 104, 105, 476
Vasopressin (Sec ADH) 497, 536 deficiency 476
Verdoglobin 462 Xenobiotic(s) 554, 555, 557
Very long chain fatty acid(s) VLCFA 327,
T
344
Vinblastine 153 YY peptide 322
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