PROTEINS

HYDROPHOBIC AMINO ACIDS

- greek "proteios" = of great importance - common: • Aliphatic HC side chain
- Organic: most abundant organic molecules in living systems • Aromatic side chain
- AMINO ACIDS - building blocks of proteins - Proline - aliphatic side chain turns back to bond with amino
- has 21 types group making it look like a ring but it is NOT.
- sequencing affects the structure and
function of a protein molecule
- has the most diverse range of functions due to various amino
Phobia of VAMP GIrL
acids
V - Valine G - Glycine
PROTEIN TYPES AND FUNCTIONS: A - Alanine I - Isoleucine
Enzymes - speed up chemical reaction rate M - Methionine (T)r - Tryptophan
- biologic catalysts P - Proline, Phenylalanine L - Leucine
- Ex:
• Digestive: amylase, lipase, pepsin, trypsin HYDROPHILIC AMINO ACIDS
• Synthase (enzymes that create): ligase - Polar-Neutral Amino Acids
Transport - carry materials from one place to another - common: OH/hydroxyl group side chain
- Ex: - no charge = neutral
• Albumin - simple protein - Cysteine - sulfhydryl group = disulfide bond
- general transporter Come Take GlAsS
• Transferrin - carries/binds iron C - Cysteine G -Glutamine
• Hemoglobin - carries oxygen T - Threonine, Tyrosine A - Asparagine
Defense - protection from antigens S - Serine
- Ex: Immunoglobulin/Antibodies
HYDROPHILIC POSITIVELY-CHARGED AMINO ACIDS
Structure - construction of different structures
- Polar-Basic Amino Acids
- for support
- common: amino group side chain
- Ex: Collagen and Keratin
Regulatory - controls cell functions Last Action Hero / LAH
- coordinates activities L - Lysine
- Ex: A - Arginine Lysine
• Hormones: insulin - metabolism of glucose H - Histidine
Contractility - allows movement of all forms
HYDROPHILIC NEGATIVELY-CHARGED AMINO ACIDS
- Ex: Actin and Myosin
- Polar-Acidic Amino Acids
Nutritional - provides nourishment necessary for development - common: carboxyl group side chain
- Ex: Albumin and Casein (from milk)
___________________________________________________ GlAss
G - Glutamic Acid
AMINO ACIDS A - Aspartic Acid Glutamic Acid
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PEPTIDE BOND
- linkage between carboxyl group of 1 amino acid and the
amino group of another amino acid
- fundamental bond of a protein molecule
- covalent bond - sharing of electrons of molecules
- formed through dehydration synthesis

Essential Amino Acids Nonessential Amino Acids

- cannot be made by the body - can be made by the body, ___________________________________________________
- must come from dietary source it is not essential to the diet
PROTEIN STRUCTURE
PTV TIM HiLL Sa Tyrone PArC GAGAGA
Primary Structure - 2D
P - Phenylalanine S - Serine - simplest level; one dimensional
T - Threonine T - Tyrosine - sequencing (arrangement or order) of amino acids in a
V - Valine P - Proline polypeptide chain
T - Tryptophan Ar - Arginine - sequence is determined by the DNA
I - Isoleucine C - Cysteine
M - Methionine G - Glutamic acid Change in Change in overall
Change in DNA amino acid structure and
Hi - Histidine A - Aspartic acid
sequence function of CHON
L - Leucine G - Glutamine
L - Lysine A - Asparagine
G - Glycine
A - Alanine
 - acts as a safety pin that stabilizes and
holds the foldings together

*ALL BONDS AND FOLDS ARE DUE TO THE R-GROUPS INTERACTIONS*

*Hemoglobin A - normal hemoglobin Quaternary Structure - 3D

- most potent in carrying oxygen - final level; most complex of all protein structure
Hemoglobin S - people with sickle-cell anemia due to substitution of - more than 1 polypeptide chain unlike other 3 levels
supposed to be glutamic acid by valine - Hb - 4 polypeptide chains (tetramer)
- vaso occlusion - happens when sickle cells are not - the interaction of all subunits (polypeptide chains) together
able to pass through small
capillaries
Secondary Structure - 2D
- local folded structures that form due to the interaction
between atoms of the backbone
- does NOT include the interaction with the side chain
- 2 CONFORMITIES: (possible due to H bonds)

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Fibrous Proteins
- long, narrow rod
- tough, insoluble and composed of fibers and sheets
• Alpha Helix - Ex: Collagen, Keratin, Elastins
- looks like a folded ribbon Globular Proteins
- H bond: between carbonyl group of 1 amino acid and - More or less spherical
hydrogen of amino group of another that is - water-soluble
4th down the chain (1 and 4 will bond together) - have chains folded into compact shapes
- R-group protruding is free to interact with others - Ex: Hemoglobin, Myoglobin, Insulin, Immunoglobulin
• Beta Pleated Sheet
- H bond: between carbonyl group of 1 amino acid and Dietary Proteins
hydrogen of amino group of another that is - Simple - composed of purely amino acid residues
- R-group protruding is free to interact with others - Ex: Albumin, Ribonuclease (124 amino acids)
- Conjugated - protein incorporated with 1 or more non-
Tertiary Structure - 3D amino acid units
- polypeptide chain within its regions of 2° structure further - Ex: Glycoproteins, Lipoproteins, Metalloproteins
folds itself - Complete - derived from animal sources
- primarily due to interactions between the R groups of the - provides all essential and non-essential amino acids
amino acids that make up the protein - Incomplete - derived from vegetable sources
- Ionic bond, hydrogen bond, dipole-dipole, London dispersion - lacks sufficient amount of 1 or more essential
- has hydrophobic interaction - amino acids containing amino acids
hydrophobic R-groups will
be tucked deep within
protein molecule
- Disulfide bonds - exists between thiol/sulfhydryl groups of
or linkage cysteines
 PHYSICAL AND CHEMICAL PROPERTIES OF
PROTEINS ISOELECTRIC FOCUSING
PROTEINS - separation based on isoelectric point
- generally tasteless
- mostly colorless
- insoluble in fat solvents
- has varied degree of solubility in water, salt solutions, dilute
acids and alkalis
- mostly high molecular weight
- mostly non-diffusible colloid solutions
- reactive and highly specific
- most are labile, readily modified in solution with alterations
in pH, UV, heat and organic solvents
- AMPHOTERIC
AMPHOTERISM
- ability of molecule to act either as an acid or base
depending on the pH of the medium
- In an alkaline pH, protein acts as an acid by donating H+ to
the solution

- In an acidic pH, protein acts as a base by accepting H+ from

the solution

ISOELECTRIC POINT (pI) ___________________________________________________

- pH at which amino acids have become zwitterions, carrying
DIFFERENT PROTEIN MOLECULES UNDER
NO or NEUTRAL electric charge
- Zwitterions - has opposite equal + and - charges on each INVESTIGATION UNDER MT
end, making it neutral Hemoglobin
- when a protein is at its isoelectric point, there is NO Immunoglobulin/Antibodies
movement of molecules along the support medium - produced by Plasma cells
ELECTROPHORESIS - highly specific
- separation of molecules based on the ratio of charge to size - has a memory
- macromolecules have different mobilities based on their - can recognize "self" from "non-self"
size, shape and charge - IgM - pentamer
- Sodium Dodecyl Sulfate Polyacrylamide Gel (SDS-PAGE) - IgA - dimer
• most commonly used gel - IgD, IgE, and IgG - monomer
• Alkaline - pH 8.6, making the proteins negatively-charged - structure of antibodies are important for their optimum
• samples are put near cathode the so molecules will race function
to anode
SDS
• provides anions to break down covalent bonds in proteins
Polyacrylamide Gel
• support medium where the proteins move along
- HOW IS IT DONE?
1. Gel as support medium
2. Samples are placed on the gel
3. Controlled electric current is applied for molecules to
separate. Molecules will then move according to their
size, shape and charge.
*Bigger = slower migration
* Smaller = faster migration
4. Staining the gel to reveal locations of proteins
 Reactants Products
*Reactants MUST interact with each other with ACTIVATION
ENERGY to produce products*
PROTEIN DENATURATION ACTIVATION ENERGY
Denaturation - the energy necessary for the reaction to start
- Net effect of alterations in the biological, chemical, and - MUST be overcome to create a reaction
physical properties of the protein by mild disruption of its - included in ALL chemical reactions
structures, usually the secondary and tertiary structures
- NOT strong enough to break peptide bonds, thus, the Energy barrier
primary structure (amino acid sequence) remains the same
after denaturation
- often reversible because the primary structure is conserved.
This is achieved upon removal of denaturing agent
Energy barrier
- WHEN IRREVERSIBLE: total loss of function of the protein
molecule
FACTORS CAUSING PROTEIN DENATURATION:
Transition
Heat - disrupts H bonds and non-polar hydrophobic stage
interactions
- increases kinetic energy, causing molecules to vibrate
rapidly that the bonds are disrupted Transition stage - where there is conversion of substrates to
products
pH - disrupts salt bridges (electrostatic interaction)
- + and - ions holding the salt bridge in the protein *If enzyme is absent, the Activation Energy is higher*
molecule are replaced by the + and - ions when acidic or *If enzyme is present, the Activation Energy is lower*
basic solutions are introduced to the protein molecule ENZYME STRUCTURE:
Heavy Metals - disrupts salt bridges in protein molecules
- Heavy metal salts replace + and - ions holding
the salt bridge in a protein molecule
- disrupts disulfide bonds
- have high affinity for sulfur
Reducing Agents - disrupts salt bridges in protein molecules
- disulfide bond is formed by oxidation of
-SH groups on cysteine Apoenzyme - protein portion
- reducing agents then split disulfide bonds - made up of amino acids
apart by adding hydrogen atoms Cofactor - nonprotein portion
Mercaptoethanol - disrupts disulfide bonds - activates enzyme
- monomers of IgM and IgA are held Holoenzyme = Apoenzyme + Cofactor
together by disulfide bons forming - complete enzyme structure
pentamers and dimers respectively - conjugated enzyme
- degrade the pantemeric structure of IgM *NOT ALL enzymes need a cofactor*
and dimeric structure of IgA to yield
monomers
Alcohol - disrupts H bonding in the secondary structure
- H bonding in the tertiary structure is disrupted by
addition of another alcohol
Detergent - disrupts hydrophobic interactions
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ENZYMES
Enzymology - study of enzymes
• Activity of Enzymes Active Site - where substrate interacts with a particular charged
• Chemical reactions on enzyme catalyses amino acid residue of the enzyme
• Clinical Use - ONLY substrate can bind to it; specific
Pineapple - enzyme Bromelain Allosteric Site - cavities or sites other than active site where
Papaya - Papain regulator molecules bind to
ENZYMES - where cofactors, inhibitors, etc bind
- biologic catalysts DEFINITION OF TERMS:
- undergo chemical change after reaction Isoenzyme
- NOT consumed during the reaction - enzymes that catalyzes same reaction but different in forms
- NEVER runs out during reaction - DIFFERENCE: physical properties, electrophoretic mobility,
- found in all body tissues solubility and resistance to activation
- act as markers of disease - Ex: LD
- significantly appears in serum/plasma:
• following cellular injury
• when cells are degraded
• during certain physiologic processes

A + B P + Q
Cofactor Enzyme Commision (E.C.) Classification
- non-protein molecule that is necessary for enzyme activity Oxidoreductase
- has the ability to change conformity of the enzyme to help - an enzyme that catalyzes an oxidation–reduction reaction
bind the substrate to it - requires a coenzyme that is oxidized or reduced as the
- can be: substrate is reduced or oxidized.
• Coenzyme - organic - usually utilizes NADP or NAD+ as cofactors
- Ex. Vit. B6 - Loss of 2 H+ atoms by a molecule is an indication of oxidation
• Activator - inorganic - Ex. Lactate dehydrogenase - removes hydrogen atoms from
- mostly metals a molecule
Zymogen
- an inactive substance form of an enzyme
- activated by another enzyme or environmental factors
- Ex. pepsinogen
- suffix -gen into -in
Transferase
- an enzyme that catalyzes the transfer of a functional group from
one molecule to another OTHER THAN H+
- Major subtypes:
• Transaminase - catalyzes the transfer of an amino group from
one moleculeto another.
• Kinases - catalyze the transfer of a phosphate group from
ATP to give ADP and a phosphorylated product (a
product containing an additional phosphate group)

NOMENCLATURE OF ENZYMES:
1. Substrate + -ase
Hydrolase
- Lipid → Lipase
- an enzyme that catalyzes a hydrolysis reaction in which the
- Ester → Esterase
addition of H2O molecule to a bond causes the bond to break.
- Protein → Protease
- Hydrolysis reactions are central to the process of digestion.
2. Reaction it catalyzes - Carbohydrases effect the breaking of glycosidic bonds
- Oxidation → Oxidase in oligo- and polysaccharides, proteases effect the breaking of
- Reduction → Reductase peptide linkages in proteins; lipases effect the breaking of ester
- Hydrolysis → Hydrolase linkages in triacylglycerols
- Removal of H+ → Dehydrogenase - Ex. Phosphatase - removes phosphate groups from biological
- Removal of Carboxyl group → Decarboxylase compounds
- Transfer of inorganic phosphate → Kinase .
3. Enzyme Commision (E.C.) Nomenclature
- Numerical code → E.C.3.2.1.1.
1st digit: CLASS: 1. Oxidoreductase
2. Transferase Lyase
3. Hydrolase - an enzyme that catalyzes the addition of a group to a double bond
4. Lyase or the removal of a group to form a double bond in a manner that
5. Isomerase does NOT involve hydrolysis or oxidation.
6. Ligase - breaking bonds between C-C, C-N, and C-O
2nd digit: SUBCLASS - Ex. Dehydratase - removal of water components from a double bond
3rd & 4th digit: SUBSUBCLASS and SERIAL NUMBER Hydratase - addition of components of water to a double bond.
- Systematic Naming → 1,4-D-Glucan Glucanohydrolase Decarboxylase- removal of CO2
- Recommended Name → α-Amylase

Isomerase
- an enzyme that catalyzes the isomerization(rearrangement
of atoms) of a substrate in a reaction, converting it
into a molecule isomeric with itself.
- transfer within the same molecule
- ONLY 1 reactant and 1 product in reactions
- Ex. Mutase - catalyze intramolecular group transfers
 - reacts w/ 1 particular optical isomer ONLY
- Ex: Glucose Oxidase

Ligase Type Reaction Type Examples

- an enzyme that catalyzes the bonding together of 2 molecules Absolute Catalyzes 1 type of reaction Urease catalyzes only
into 1 with the participation of ATP. for 1 substrate hydrolysis of urea
- ATP - required because such reactions are generally Group Catalyzes 1 type of reaction Hexokinase adds
energetically unfavorable and they require the for similar substrate phosphate group to
simultaneous input of energy obtained by a hydrolysis hexoses
reaction in which ATP is converted to ADP Linkage/ Catalyzes 1 type of reaction Chymotrypsin catalyzes
- Ex. DNA Ligase Bond for a specific type of bond the hydrolysis of
peptide bonds
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ENZYME REACTION THEORIES
Lock and Key Model
- active site in the enzyme has a fixed, rigid geometrical conformation.
- ONLY substrates with a complementary geometry can bind

Induced-Fit Model
- E shape is too restrictive for the action of many other enzymes.
- many enzymes have flexibility in their shapes. They are not
rigid and static; there is constant change in their shape.
- allows for small changes in the shape or geometry of the
active site of an enzyme to accommodate a substrate.
- adapts to accept the incoming substrate

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ENZYME KINETICS
- studies the speed of enzyme activity
Michaelis Menten Model
- overall reaction for an enzyme-catalyzed reaction:
E+S ES complex E + Product
- [E] Kinetics = rate of enzyme activity
- STEADY STATE ASSUMPTION
• rate of E & S binding = rate of product formation

E+S * S rate of reaction

E+P
Substrate Concentration

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ENZYME REACTIONS
SPECIFICITY
Absolute Specificity - FIRST ORDER KINETICS/REACTION
- combines with only 1 substrate • Rate of enzyme activity is DEPENDENT on substrate concentration
- catalyzes 1 specific reaction
Group Specificity
- combines w/ ALL substrates containing particular chemical group
- Ex: Phosphate Ester More enzyme molecules can react
Bond Specificity with more substrate molecules
- specific to a chemical bond = reaction rate increases
Stereoisometric Specificity
 protonation or deprotonation
Substrate Concentration of groups on the substrate.
- changes in pH may denature the enzyme
Enzyme Location Substrate Optimum pH
ZERO ORDER KINETICS/REACTION
Pepsin stomach Peptide bonds 1.5-2.0
• Rate of enzyme activity is INDEPENDENT on substrate
concentration Lactase GI tract lactose 6.0
• Enzyme activity is measured REGARDLESS of substrate Sucrase Small intestine Sucrose 6.2
concentration Amylase Pancreas Amylose 6.7-7.0
• # of enzymes is constant no matter the substrate concentration Urease Liver urea 7.0
Trypsin Small intestine Peptide bonds 7.7-8.0
Lipase Pancreas Lipid (ester bonds) 8.0
First Zero order at plateau
order
Arginase Liver arginine 9.7

Temperature
- Optimum temperature - temperature at which an
enzyme exhibits maximum activity
- kinetic energy - higher temp = molecules are
moving faster and colliding more frequently;
collisions between S molecules and E.
Vmax = limit of enzyme reaction because # of enzymes is - If temperature increases beyond a certain point
constant no matter the substrate concentration = disruptions in the tertiary structure of the
= depend on enzyme concentration enzyme; denaturation is occurring
First order reaction = gives relationship of rate and S - usually 37°C
concentration - Denaturation at 40-50°C
Km = Michaelis Constant - Assay temperatures: 25°C, 30°C, 37°C
= concentration of substrates when the reaction reaches half
Substrate Concentration
of Vmax.
- concentration of an enzyme is kept constant
= E+S affinity (inverse)
and concentration of substrate is increased
- Km E+S affinity
- As substrate concentration increases, the
- Km E+S affinity
point is eventually reached where enzyme
A small Km indicates high affinity since it means the reaction can capabilities are used to their maximum extent.
reach half of Vmax in a small number of substrate concentration. The rate remains constant from this point on.
Enzyme Concentration
- If the amount of substrate present is kept
constant and the enzyme concentration is
increased, the reaction rate increases
because more substrate molecules can be
accommodated in a given amount of time
Cofactors
- non-protein molecules that must bind to particular enzymes to
___________________________________________________ create a reaction
SUBSTRATE CONCENTRATION - coenzymes - serve as secondary substrate
First Order Reactions - Ex: Ca, Fe, Mg, Mn, Zn, K, Br, Cl
-A P
Inhibitors
- ONLY 1 reactant
- interfere with enzyme reactions (slows or stop catalytic reactions)
- direct relationship of enzyme concentration and
- Competitive inhibitor
substrate concentration
• reversible; complex breaks up with time
- Rate is proportional to concentration of 1 reactant
• molecule sufficiently resembling an enzyme substrate in shape
- Rate of reaction is proportional to rate of disappearance
and charge distribution that it can compete for occupancy
of reactants OR rate of appearance of products
of the enzyme’s active site
Second Order Reactions • roughly the same shape as substrates, making it fit on active site,
-A+B P substrate is then unable to bind to enzyme and NO reaction occurs
- 2 reactants • often a product of enzyme-catalyzed reaction
- Rate is proportional to product of concentration of 2 • Competitive inhibition can be reduced by simply increasing
reactants the substrate concentration. Reverse is also true
- direct relationship of enzyme concentration and substrate • increase in Km of the enzyme, but leaves Vmax unaffected
concentration - Non-competitive/Allosteric inhibitior
Zero Order Reactions • reversible
- Rate of reaction DEPENDS on concentration of enzyme OR on • molecule that decreases enzyme activity by binding to a site
some factors OTHER THAN concentration of molecular species on an enzyme other than the active site.
undergoing reaction • often a product of enzyme-catalyzed reaction
- In ENZYME ACTIVITY: even if substrate is doubled, reaction will • binds to allosteric site and changes active site shape =
NOT increase any further substrate can't bind to the enzyme
____________________________________________________________________________________________________________ • decrease Vmax and will not affect Km.
Factors affecting Enzyme Reactions • Ex: the heavy metal ions Pb2+, Ag+ and Hg2+
pH (usually 7.0-8.0) - Uncompetitive
- Optimum pH - pH at which an enzyme • only bind to the enzyme when substrate is bound to the enzyme
exhibits maximum activity • decrease both Vmax and Km
- depends on enzyme
- A variation from normal pH can also
affect substrates, causing either
 - Prostate Gland - Prostatitis & Prostatic Carcinoma
- RBC
- Platelets

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Clinically Significant Enzymes
OXIDOREDUCTASE
- Lactate Dehydrogenase (LD/LDH)
• LD-1 - heart • LD-3 • LD-5
• LD-2 - heart • LD-4
- Glucose-6-Phosphate Dehydrogenase (G-6-PD)
• Red Blood cells
TRANSFERASE
- Gamma-Glutamyltransferase (GGT)
• transfer of gamma-glutamyl residue FROM gamma-glutamyl
peptides TO amino acids, H2O, and other small peptides
• Glutathione - glutamyl donor
Glutathione + Amino Acid Glutamyl - Peptide + L-cysteinylglycine
- Aspartate Aminotransferase (AST)
• Glutamate oxaloacetate transaminase (GOT)
• Heart, Liver
• catalyzes the reversible transfer of an α-amino group
between aspartate and glutamate

- Creatine Kinase (CK)

• CK-1 - Brain (CK-BB)
• CK-2 - Myocardium (CK-MB)
• CK-3 - Skeletal Muscles (CK-MM)
HYDROLASE
- Amylase (AMY)
• Salivary Gland (S-type)
• Pancreas (P-type)
- Lipase (LPS)
• Pancreas
- Cholinesterase (ChE)
• hydrolysis of these cholinergic neurotransmitters
• True ChE - Synapse of the nerve (AChE)
• Pseudo ChE - RBC (PChE)
- Phosphatase
• water to cleave a phosphoric acid monoester
into a phosphate ion and an alcohol

○ Alkaline Phospatase
- Bone
• Osteoblasts - involved in production of bone matrix
- Placenta
- Kidney
- Intestine
- Liver
• Sinusoidal and bile canalicular membranes
- Spleen
○ Acid Phosphatase