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CHEMISTRY AND BIOCHEMISTRY OF PROTEINS II

METABOLISM OF AMINO ACIDS

• Proteins are the most abundant organic compounds and constitute a major part
of the body dry weight (10-12kg in adults).
• Perform a wide variety of structural and dynamic (enzymes, hormones, clotting
factors, receptors) functions.
• Proteins are nitrogen containing macromolecules consisting of L-α -
amino acids as the repeating units or chains connected by peptide bonds.
A peptide bond is an amide bond formed between amino acids by the
condensation of –NH2 and -COOH, releasing H2O.
• There are 20 different types of amino acids that constitute proteins, and the
sequence of amino acids determines the structure and properties of the resulting
protein.
• Of the 20 amino acids found in proteins, half can be synthesized by the body
and half are supplied through diet.
• The proteins on degradation release individual amino acids. Each amino acid
undergoes its own metabolism and performs specific functions.

• Amino acid metabolism refers to the sum of all chemical reactions in which amino
acids are broken down and synthesized for vital processes in the body.
• Amino acids can be divided into two types: essential and non-essential amino acids.
• Essential amino acids are amino acids necessary for an organism's survival. Since the
organism cannot synthesize these essential amino acids by themselves, they must
obtain them from their diets. Generally, animals cannot synthesize nine amino acids
(Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine,
Tryptophan, Valine).
• Non-essential amino acids are amino acids that can be synthesized by the body. The
remaining 11 are nonessential or conditionally essential amino acids.
• Animals have seven conditionally essential amino acids (Arginine, Cysteine,
Glutamine, Glycine, Proline, Tyrosine, Serine) that can be synthesized and are usually
not required in the diet. However, they are essential components of the diet for specific
populations that cannot synthesize them in adequate amounts.
• There are four “nonessential” amino acids (Alanine, Asparagine, Aspartate,
Glutamate) that can be synthesized and, thus, are not required in diet.

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• Amino acid degradation produces

ammonium (NH4+) and a carbon
skeleton. The NH4+ is removed either
by synthesis of nitrogen-containing
compounds such as nucleotides, or
excreted in the form of urea.
• The carbon skeletons of amino acids
can be converted into TCA cycle
intermediates, which are used either to
generate ATP through oxidative
phosphorylation or provide the
precursors for fatty acid synthesis and
gluconeogenesis

• Some amino acids serve as precursors for the synthesis of many biologically
important compounds. Certain amino acids may directly act as neurotransmitters
(e.g glycine, aspartate, glutamate).
Amino Acid Pool
• About 100g of free amino acids which represent the amino acid pool of the body.
Glutamate and glutamine together constitute about 50%, and essential amino acids
about 10% of the body pool (100g). The concentration of intracellular amino acids
is always higher than the extracellular amino acids. Amino acids usually enter the
cells against a concentration gradient.
Sources of Amino Acid
• Turnover of body intake of dietary protein and the synthesis of non-essential amino
acids contribute to the body amino acid pool.
• Protein turnover:
• The proteins in the body is in a dynamic state.
• About 300-400g of protein per day is constantly degraded and synthesized, which
represents body protein turnover.

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METABOLIC PATHWAYS THAT PRODUCE

NONESSENTIAL AMINO ACIDS
• Animals lack the enzymes to generate the essential amino acids, thus, these
amino acids must be obtained from the diet. The structures of the essential
amino acids, generally, are more complex than the nonessential amino acids.
• Essential amino acids require a significantly greater number of enzymatic
reactions for synthesis and are found in plants and lower organisms. Tyrosine
and arginine are conditionally essential. Tyrosine is derived from the essential
amino acid phenylalanine by the enzyme phenylalanine hydroxylase.
Endogenous tyrosine production is dependent on dietary phenylalanine, and a
significant portion of tyrosine is obtained from diet. A small amount of arginine
can be generated from argininosuccinate in the urea cycle.
• Animals can synthesize the conditionally essential and nonessential amino
acids using glycolytic and TCA cycle intermediates. The glycolytic
intermediate 3-phosphoglycerate generates serine and glycine.

Control of protein turnover

• The turnover of the protein is influenced by many factors.
• A small polypeptide called ubiquitin (m.w.8,500) tags with the
protein and facilitates degradation.
• Certain proteins with amino acid sequence proline, glutamine, serine
and threonine are rapidly degraded
• Dietary protein: There is a regular loss of nitrogen from the body due
to degradation of amino acids. About 30-50g of protein is lost every
day. This amount of protein is supplied through diet to maintain
nitrogen balance. There is no storage form of amino acids in the
body. Excess intake of amino acids is oxidized to provide energy.

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• Proteins function as enzymes, hormones, immunoproteins,

contractile proteins etc. Many important nitrogenous compounds
(porphyrins, purines, pyrimidines, etc) are produced from the
amino acids. About 10-15% of body energy requirements are met
from the amino acids. The amino acids are converted into
carbohydrates and fats.

Catabolism of Amino Acids Occur in 4 stages

• Transamination
• Oxidative Deamination
• Ammonia Transport
• Urea Cycle

Transamination
• The transfer of an amino (-NH2) group from an amino acid to a ketoacid, with the
formation of a new amino acid and a new keto acid.
• Catalysed by a group of enzymes called transaminases (aminotransferases) that uses
pyridoxal phosphate (PLP) as a co-factor.
• The liver, Kidney, Heart, and Brain have adequate amount of these enzymes.

Salient Features of Transamination

• All transaminases require PLP.
• No free NH3 liberated, only the transfer of amino group.
• Transamination is reversible.
• There are multiple transaminase enzymes which vary in substrate specificity. AST and ALT
make a significant contribution for transamination.
• Transamination is important for redistribution of amino groups and production of non-
essential amino acids.
• It diverts excess amino acids towards the energy generation.
• Amino acids undergo transamination to finally concentrate nitrogen in glutamate.

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• Glutamate undergoes oxidative deamination to liberate free NH3

for urea synthesis.
• All amino acids except, lysine, threonine, proline and
hydroxyproline participate in transamination.
• It involves both anabolism and catabolism, since – reversible.

AA + α- KG ketoacid + Glutamate
Alanine + α- KG Pyruvate + Glutamate
Aspartate + α- KG Oxaloacetetae + Glutamate

Mechanism of Transmission
Step:1
• Transfer of amino group from AA1 to the coenzyme PLP to form
pyridoxamine phosphate.
• Amino acid1 is converted to Keto acid2.

Step:2
• Amino group of pyridoxamine phosphate is then transferred to a
keto acid1 to produce a new AA 2 and enzyme with PLP
regenerated.

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Transamination reaction

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Clinical Significance
• Enzymes, present within cell, released in cellular damage into blood.
• ↑ AST - Myocardial Infarction.
• ↑ AST, ALT – Hepatitis, alcoholic cirrhosis.
• Muscular Dystrophy.

Trans-deamination
• The amino group of most of the amino acids is released by a coupled reaction,
transdeamination.
• Transamination followed by oxidative deamination.
• Transamination takes place in the cytoplasm.
The amino group is transported to liver as glutamic acid, which is finally
oxidatively deaminated in the mitochondria of hepatocytes.

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Deamination
• The removal of amino group from the amino acids as NH3 is deamination.
• Deamination results in the liberation of ammonia for urea synthesis.
• The carbon skeleton of amino acids is converted to keto acids.
• Deamination may be either oxidative or non-oxidative
• Only liver mitochondria contain glutamate dehydrogenase (GDH) which
deaminates glutamate to α-ketoglutarate and ammonia. It needs NAD+ as co-
enzyme.
• GDH is an allosteric enzyme. It is activated by ADP and inhibited by GTP.
Oxidative deamination is the liberation of free ammonia from the amino group
of amino acids coupled with oxidation.
Mostly in liver and kidney. Oxidative deamination is to provide NH3 for urea
synthesis and α-keto acids for a variety of reactions, including energy
generation.

Role of Glutamate Dehydrogenase

• Glutamate is a 'collection centre' for amino groups.
• Glutamate rapidly undergoes oxidative deamination.
• Catalysed by GDH to liberate ammonia.
• It can utilize either NAD+ or NADP+.
• This conversion occurs through the formation of an α-iminoglutarate

Oxidative of glutamate by GDH

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Metabolic Significance
• Reversible Reaction
• Both Anabolic and Catabolic.
• Regulation of GDH activity:
• Zinc containing mitochondrial, allosteric enzyme.
• Consists of 6 identical subunits.
• Molecular weight is 56,000.

Allosteric regulation
• GTP and ATP – allosteric inhibitors.
• GDP and ADP - allosteric activators.
• ↓ Energy - ↑ oxidation of amino acid.
• Steroid and thyroid hormones inhibit GDH.

Amino Acid Oxidases

• L-amino acid oxidase and D-amino acid oxidase.
• Flavoproteins and Cofactors are FMN and FAD.
• Act on corresponding amino acids to produce α-keto acids and NH3
• It occurs in the Liver, kidney, Peroxisomes.
• Activity of L-amino acid oxidase is low and plays a minor role in amino
acid catabolism.
• L-amino acid oxidase acts on all amino acids, except glycine and
dicarboxylic acids.
• Activity of D-amino oxidase is high than that of L-amino acid oxidase
• D-amino oxidase degrades D-amino acids in bacterial cell wall.

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Amino acid oxidases

Fate of D-amino acids

• D-amino acids are found in plants and microorganisms. They are not
present in mammalian proteins.

• D-amino acids are taken in the diet/bacterial cell wall, absorbed from
gut. D-amino acid oxidase converts them to the respective α-keto
acids.

• The α-ketoacids undergo transamination to be converted to L-amino

acids which participate in various metabolic pathways. Keto acids
may be oxidized to generate energy or serve as precursors for glucose
and fat synthesis.

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Non-oxidative Deamination
• This is a direct deamination without oxidation.
• Amino acid Dehydratases:
• Serine, threonine and homoserine are the hydroxy amino acids.
• They undergo non-oxidative deamination catalyzed by PLP-dependent
dehydratases

Amino acid Desulfhydrases

• Cysteine and homocysteine undergo deamination coupled with desulfhydration
to give keto acids.

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UREA CYCLE
• The urea cycle is the first metabolic pathway to be elucidated. The cycle is
known as Krebs–Henseleit urea cycle.
• Ornithine is the first member of the reaction, it is also called as Ornithine cycle.
Urea is synthesized in liver and transported to kidneys for excretion in urine.
• The two nitrogen atoms of urea are derived from two different sources, one
from ammonia and the other directly from the amino group of aspartic acid.
Carbon atom is supplied by CO2.
• Urea is the end product of protein metabolism (amino acid metabolism).
• Urea accounts for 80-90% of the nitrogen containing substances excreted in
urine. Urea synthesis is a five-step cyclic process, with five distinct enzymes.
The first two enzymes are present in mitochondria while the rest are localized
in cytosol.

Glutamine and glutamate generate two

nitrogen molecules for the urea cycle. Urea
contains two nitrogen molecules.
Glutaminase generates NH4 and glutamate.
+

Subsequently, glutamate can be converted

into α-ketoglutarate to liberate another
NH4+ by glutamate dehydrogenase.
Carbamoyl phosphate synthetase (CPS1)
uses NH4+ , HCO3-, and ATP as substrates to
generate carbamoyl phosphate, which
provides the first source of nitrogen
molecules for urea generation. Amino acids,
such as alanine, can also be converted into
glutamate through aminotransferases. Next,
the aspartate aminotransferase converts
glutamate into aspartate, which feeds into
the urea cycle to provide the second source
of nitrogen molecules for urea production

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Step 1: Formation of Carbamoyl phosphate

• Carbamoyl phosphate synthase I (CPS I) of mitochondria catalyses the
condensation of NH4+ ions with CO2 to form carbamoyl phosphate.
• This step consumes two ATP and is irreversible. It is a rate-limiting.
• CPS I requires N-acetylglutamate (NAG) for its activity.
• Meanwhile, Carbamoyl phosphate synthase II (CPS II) involved in pyrimidine
synthesis and it is present in cytosol. It accepts amino group from glutamine and
does not require N-acetylglutamate for its activity.

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Carbamoyl phosphate (CPS)

CPS I CPS II

• Mitochondria • Cytosol

• Uses NH3 • Uses Glutamine

• Urea cycle • Pyrimidine biosynthesis

• Activated - N-acetylglutamate • Inhibited - CTP

Step 2: Citrulline formation

• The second reaction is also mitochondrial. Citrulline is synthesized from
carbamoyl phosphate and ornithine by ornithine transcarbamoylase.
• Ornithine is regenerated and used in urea cycle. Ornithine and citrulline are
basic amino acids (but never found in protein structure due to lack of
codons).
• Citrulline is transported to cytosol by a transporter system. Citrulline is
neither present in tissue proteins nor in blood; but it is present in milk.

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Step 3: Argininosuccinate formation

• Citrulline that is exported to the cytosol condenses with aspartate to form

argininosuccinate by the enzyme argininosuccinate synthetase.
• The second amino group of urea is incorporated at this stage. This requires
ATP and it is cleaved to AMP and PPi
• 2 high energy bonds are required which are immediately broken down to
inorganic phosphate (Pi).

Step 4: Arginine formation

• The enzyme argininosuccinase or argininosuccinate lyase cleaves

argininosuccinate to arginine and fumarate (an intermediate in TCA cycle)
• Fumarate provides connecting link with TCA cycle or gluconeogenesis.
• The fumarate is converted to oxaloacetate via fumarase and malate
dehydrogenase (MDH) and transaminated to aspartate. Aspartate is
regenerated in this reaction.

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Step 5: Urea formation

• Arginase is the 5th and final enzyme that cleaves arginine to yield urea and
ornithine. Ornithine is regenerated, enters mitochondria for its reuse in the urea
cycle. Arginase is activated by Co2+ and Mn2+
• Ornithine and lysine compete with arginine (competitive inhibition). Arginase is
mostly found in the liver, while the rest of the enzymes (four) of urea cycle are
also present in other tissues.
• Arginine synthesis may occur to varying degrees in many tissues. But only the
liver can ultimately produce urea

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The overall reaction may be summarized as:

• NH3 + CO2 + Aspartate → Urea + fumarate
• 2ATPs are used in the 1st reaction.
• Another ATP is converted to AMP + PPi in the 3rd step, which is equivalent
to 2 ATPs.
• The urea cycle consumes 4 high energy phosphate bonds.
• Fumarate formed in the 4th step may be converted to malate.
• Malate when oxidised to oxaloacetate produces 1 NADH equivalent to 2.5
ATP.
• So net energy expenditure is only 1.5 high energy phosphates.
• The urea cycle and TCA cycle are interlinked and it is called as "urea
bicycle“.

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Significance of Urea cycle

• Toxic ammonia is converted into non-toxic urea.
• Synthesis of semi-essential amino acid-arginine.
• Ornithine is precursor of Proline, Polyamines.
• Polyamines include putrescine, spermidine, spermine.
• Polyamines have diverse roles in cell growth and proliferation
Regulation of Urea cycle
• Carbamoyl phosphate synthase (CPS-I) is the rate limiting enzyme in urea
cycle. CPS-I is allosterically activated by N-acetylglutamate (NAG) that is
synthesized from glutamate and acetyl CoA by NAG synthase and degraded by
a NAG hydrolase.
• The rate of urea synthesis in liver is correlated with the concentration of N-
acetylglutamate.

NAG formation and degradation

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• High concentrations of arginine increase NAG level. The consumption of a

protein-rich meal increases the level of NAG in liver, leading to enhanced urea
synthesis.
• CPS-I and GDH are present in mitochondria. They coordinate with each other
in the formation of NH3 and its utilization for carbamoyl phosphate synthesis.

Urea disposal
• Urea produced in the liver freely diffuses and is transported in blood to kidneys and
excreted. A small amount of urea enters the intestine where it is broken down to
CO2 and NH3 by the bacterial enzyme urease.
• This ammonia is either lost in the feces or absorbed into the blood.
Disorders of the Urea cycle
• The main function of Urea cycle is to remove toxic ammonia from blood as
urea.
• Defects in the metabolism of conversion of ammonia to urea, i.e., Urea cycle
leads to Hyperammonaemia or NH3 intoxication. Hyperammonaemia could
results from inherited disorders of urea cycle enzymes (familial
hyperammonaemia) or acquired disorders (Liver Disease, severe renal disease
(acquired hyperammonaemia).
• Ammonia toxicity can cause: increased levels of ammonia crossing the Blood
Brain Barrier, formation of glutamate, increase utilization of α-ketoglutarate,
decreased levels of α- Ketoglutarate in Brain. Since α-KG is a key
intermediate in TCA cycle, its decreased levels will impairs TCA cycle and
dcreased ATP production.

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Inherited disorders of Urea Cycle

• N-Acetylglutamate Synthase Deficiency

• Autosomal Recessive and a severe neonatal disorder with fatal consequences.
• Treatment with structural analog N-carbamoyl-L glutamate activates CPS-I.

Ornithine Transporter Deficiency (ORNT1 gene)

• Ornithine is accumulated in Cytoplasm.
• HHH syndrome – Hyper-ornithinemia, Hyperammonemia, Homocitrillinuria.
Symptoms
• Increased levels of ammonia results in slurring of speech, blurring of the
vision, convulsions, nausea, vomiting, neurological deficits, mental retardation,
coma and death

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Methionine Metabolism
• Methionine metabolism is necessary for epigenetic regulation and cysteine
production. Epigenetics is a powerful mechanism by which genes are modulated
without altering the underlying DNA code. The histone proteins that wrap DNA can
undergo modifications that make the DNA either accessible or inaccessible to
proteins that either activate or repress genes.
• One modification is methylation catalyzed by histone methyltransferase enzymes
that add methyl groups to specific residues on different histones. There are also
histone demethylases that remove the methyl groups. Methionine provides the
methyl group for many histone methyltransferases, as well as other type of
methyltransferases, including those involved in the conversion of norepinephrine to
epinephrine.
• These methyltransferases use S-adenosylmethionine (SAM). SAM is generated by
condensation of ATP and methionine catalyzed by methionine adenosyltransferase.
The methyl group (CH3) is attached to the methionine sulfur atom in SAM. During
the generation of SAM, all the ATP phosphates are lost so that only the adenosine
component is attached to methionine

• SAM is converted into S-adenosylhomocysteine (SAH) upon transferring its methyl group to
DNA or proteins. SAH is then cleaved by adenosylhomocyteinase to yield homocysteine and
adenosine. Methionine synthase can convert homocysteine back to methionine, a reaction
that requires 5-MTHF (5-methyltetrahydrofolate) as the methyl donor. The resulting THF can
undergo a series of reactions, known as the folate cycle, to generate 5MTHF.
• Dietary folate provides THF. Betaine-homocysteine S-methyltransferase can also generate
methionine by using homocysteine and betaine as substrates. Homocysteine can generate
cysteine by a series of reactions known as trans-sulfuration. Homocysteine condenses with
serine to produce cystathionine, which is subsequently cleaved by cystathionine γ-lyase (also
called cystathionase) to produce α-ketobutyrate and cysteine.
• α-Ketobutyrate is converted to propionyl-CoA and then, via a three-step process, to the TCA
cycle intermediate succinyl-CoA. The cysteine can undergo a series of reactions with
glutamate and glycine to generate the cellular antioxidant glutathione (GSH). The rate-
limiting enzyme of GSH synthesis is the generation of γ-glutamyl cysteine by glutamate-
cysteine ligase.
• It is important to note that cysteine found in the blood is typically cystine, an amino acid
formed by the oxidation of two cysteine molecules covalently linked by a disulfide bond.
Cystine can be transported into cells and readily converted into cysteine by nonenzymatic
reduction and provide another source of cysteine for GSH production. The transporter
proteins SLC7A11 or xCT transport cystine in exchange for glutamate.

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Methionine Metabolism

TYROSINE METABOLISM
• Tyrosine is the precursor for the catecholamines norepinephrine, epinephrine,
and dopamine. Tyrosine hydroxylase converts tyrosine to generate
dihydroxyphenylalanine (LDOPA), a metabolic precursor to dopamine and
dopaquinone. Tyrosine hydroxylase requires the enzyme cofactor
tetrahydrobiopterin. L-DOPA is converted to dopamine by the enzyme
aromatic amino acid decarboxylase. Dopamine is a potent neurotransmitter
that is required for numerous brain functions. Notably, patients with
Parkinson’s disease, a debilitating condition marked by motor impairment and
tremors, have few normal dopamine-producing cells in the midbrain area
called the substantia nigra.
• Thus, L-DOPA is often prescribed to patients with Parkinson’s disease to
elevate their dopamine levels. There is also accumulating evidence that too-
high levels of dopamine are observed in schizophrenia. The antipsychotic
drugs for treatment work, in part, by limiting dopamine levels. Dopamine is
also a precursor to epinephrine and norepinephrine, produced in the adrenal
medulla. Catecholamines are associated with the fight-or-flight response and
prepare the body to deal with environmental stress.

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• The effects of catecholamines are associated with the sympathetic nervous system
and increase blood pressure, heart rate, and blood glucose levels. Tyrosine is also a
precursor to the production of melanin, eumelanin, and pheomelanin by melanocytes
to provide skin and hair pigmentation. Eumelanin provide dark pigments, such as
brown or black, and pheomelanin give rise to red or yellow pigmentation. The ratio
of eumelanin and pheomelanin in melanocytes dictates skin and hair pigmentation.
People lacking the gene tyrosinase cannot generate pigments; thus, they exhibit
albinism. Given the multiple roles of tyrosine metabolism, the maintenance of
tyrosine levels within cells is vital.
• Tyrosine can be obtained from the diet or generated from phenylalanine. The enzyme
phenylalanine hydroxylase converts dietary phenylalanine to tyrosine. Genetic
defects in the phenylalanine hydroxylase gene result in the metabolic disease
phenylketonuria (PKU),in which phenylalanine accumulates significantly in the
blood. PKU patients show neurological and developmental problems because of the
high levels of phenylalanine, which generate toxic metabolites, such as
phenylpyruvate, phenylacetate, and phenyl lactate.
• PKU disease is autosomal recessive and one of the more common metabolic genetic
disorders. PKU patients are diagnosed shortly after birth as a result of a simple and
routine blood test and must be on lifelong strict phenylalanine-limited diet to limit
the buildup of phenylalanine and so prevent neurological and developmental
complications.

Tyrosine metabolism produces

neurotransmitters, catecholamines, and
melanin. Tyrosine hydroxylase converts
tyrosine to generate dihydroxyphenylalanine
(DOPA), which is converted to dopamine by
the enzyme aromatic amino acid carboxylase.
Dopamine generates norepinephrine through
dopamine β-hydroxylase.
Phenylethanolamine N-methyltransferase
converts norepinephrine to epinephrine.
Tyrosine is also a precursor for melanins
through tyrosine or DOPA oxidation to
dopaquinone by the enzyme tyrosinase.
Phenylalanine hydroxylase produces tyrosine
from phenylalanine. Genetic defects in the
phenylalanine hydroxylase gene result in the
metabolic disease phenylketonuria (PKU).
PKU patients show high levels of
phenylalanine that generate toxic metabolites,
such as phenylpyruvate, resulting in
neurological and developmental problems.

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INBORN ERRORS OF METABOLISM

• Inborn errors of metabolism occur when some enzyme involved in metabolism
is abnormal. The abnormality occurs due to a mutation in gene encoding the
enzyme The affected enzyme may be absent or deficient  Inborn errors may
occur in metabolism of all nutrients including amino acids When an enzyme is
absent or deficient, metabolism of the concerned amino acid becomes
abnormal.
• Over 50 inborn errors of metabolism of amino acids have been discovered due
to decreased synthesis of products ,accumulation of intermediates ,formation of
alternate metabolites. Many disorders result in neurological abnormalities and
mental retardation. Early diagnosis and treatment can prevent neurological
abnormalities. Generally, the treatment comprises restricted intake or exclusion
of the affected amino acid from the diet.
• Some relatively common inborn errors are: • Maple syrup urine disease
(MSUD) • Cystinuria • Phenylketonuria (PKU) Alkaptonuria •Albinism

PHENYLKETONURIA
• Phenylketonuria (PKU) is the commonest inborn error of amino acid
metabolism. It has an incidence of about 1 in 10,000 live births. It was the first
inborn error of amino acid metabolism to be treated successfully by diet
manipulation. In PKU, there is a block in the conversion of phenylalanine into
tyrosine which is then converted to phenyl pyruvate and excreted in urine.

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• PKU was first described by Asbjørn Følling, one of the first Norwegian
physicians to apply chemical methods to the study of medicine. In 1934, the
mother of two intellectually impaired children approached Følling to ascertain
whether the strange musty odour of her children’s urine might be related to their
intellectual impairment.
• The urine samples were tested for a number of substances including ketones.
When ketones are present, urine usually develops a red-brown colour upon the
addition of ferric chloride, but in this instance the urine yielded a dark-green
colour. After confirming that the unusual result was not due to any medications
and repeating the test every other day for two months, Følling proceeded with a
more detailed chemical analysis involving organic extraction and purification of
the responsible compound, and determination of its melting point.
• Følling postulated that the compound was phenylpyruvic acid. Family studies of
affected individuals led to the suggestion of an inherited recessive autosomal
trait. He published his findings and suggested the name ‘imbecillitas
phenylpyruvica’ relating the intellectual impairment to the excreted substance,
thereafter renamed ‘phenylketonuria’.

The condition is also called PAH deficiency, phenylalanine hydroxylase

deficiency, Folling’s disease, PKU.
• Autosomal recessive disorder caused by mutation in Phenylalanine
Hydroxylase (PAH) gene that is located on chromosome 12q23.2. The gene
codes for the formation of the enzyme phenylalanine hydroxylase. A carrier
does not have symptoms of the disease, but can pass on the defective gene to
the progeny.
• The common symptoms includes mental retardation, microcephaly,
hyperactivity, stunted growth, skin rashes, fair skin and blue eyes
(phenylalanine cannot transform into melanin), musty odour in breath or
urine, seizure, tremor, jerking movement in the arms and legs, behavioral and
social problems.
• Treatment: PKU can be treated with a low-protein diet and dietary
supplements. The diet must be strictly followed. If continued there is a high
chance of better physical and mental health. Administration of
tetrahydrobiopterin is recommended

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• Biochemistry of PKU: Phe exists as D and L enantiomers, and L-Phe is an essential

amino acid required for protein synthesis in humans. As with many other metabolites,
Phe concentrations are regulated to a steady state level with dynamic input and runout
flux. Persistent disturbance to the flux will eventually result in alteration of the steady
state concentrations.
• Dietary intake of Phe along with endogenous recycling of amino acid stores are the
major sources of Phe, whereas, utilisation or runout of Phe occurs via integration into
proteins, oxidation to Tyr, or conversion to other metabolite.
• The conversion of Phe to Tyr occurs by a hydroxylating system consisting of: (1)
Phenylalanine Hydroxylase (PAH), (2) the unconjugated pterin cofactor
{tetrahydrobiopterin (BH4)}, (3) enzymes which serve to regenerate BH4, namely
dihydropteridine reductase and 4α-carbinolamine dehydratase
• While the para-hydroxylation of Phe is essential for the rupture of the benzene ring, it
is not required for further metabolism of the alanine side chain. This alternative
pathway of transamination and decarboxylation leads to the formation of metabolites
such as phenylpyruvate, phenylactate, and o-hydroxyphenylacetate which are excreted
in urine. Conversion of Phe to Tyr has two outcomes.
• First, it drives the endogenous production of the non-essential amino acid, Tyr.
Second, the hydroxylation reaction is the rate limiting step for complete oxidation of
Phe to CO2 and H2O and contributes to the pool of glucose and 2-carbon metabolites.

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• Phenylalanine Hydroxylase (PAH) catalyses the stereospecific

hydroxylation of L-Phe, the committed step in the degradation of this
amino acid. Phe catabolism and PAH activity is mainly associated
with the liver, although minor activity has been demonstrated in rat
kidney.
• In humans, the PAH enzyme exists as a mixture of tetramers and
dimers; the monomer is about 50 kDa in size and is comprised of 452
amino acids. PAH requires BH4 as a cofactor, as well as molecular
oxygen for its activity.
• PAH can be divided into a number of functional domains. The
regulatory domain contains a serine residue which is thought to be
involved in activation by phosphorylation. The catalytic domain
contains a motif of 26 or 27 amino acids responsible for cofactor and
ferric iron binding. The C-terminal domain is thought to be associated
with inter-subunit binding.

Structural components of PAH. The catalytic domain of PAH contains a motif of 26 or

27 amino acids which are responsible for ferric iron and cofactor (BH4) binding.

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• PAH is regulated by a number of possible mechanisms. After a protein

meal, it is postulated that the increased Phe in the amino acid pool
causes a release of glucagon from the pancreas. Hepatic PAH is
subject to control by cAMP-dependent protein kinase and α-
andrenergic agent stimulated Ca2+/calmodulin dependent protein
kinase phosphorylation–dephosphorylation processes.
• It has been further reported that these control mechanisms influence
BH4 co-factor interaction with PAH. In addition, there is evidence that
Phe may also be able to cause a conformational change in PAH, as
well as up-regulate cAMP activity. Taken together, these mechanisms
enable fine regulation of Phe concentrations by balancing levels
sufficient for maintenance of protein biosynthesis while minimising
tissue exposure to high concentrations of Phe.

Clinical presentations of PKU

• Untreated PKU is associated with an abnormal phenotype
including growth failure, microcephaly, seizures and intellectual
impairment caused by the accumulation of toxic by-products of
Phe.
• Moreover, decreased or absent PAH activity can lead to a
deficiency of Tyr and its downstream products, including
melanin, L-thyroxine and the catecholamine neurotransmitters.

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CYSTINURIA
• Cystinuria is an inherited disease that causes stones made of the amino acid cystine
to form in the kidneys, bladder, and ureters. Inherited diseases such as cystinuria are
passed down from parents to their progeny. A defect or mutation in the genes,
SLC3A1 and SLC7A9 causes cystinuria. These genes encode for the proximal
tubule dibasic amino acid transporter which facilitates reabsorption of cysteine from
tubular fluid
• To come down with cystinuria, a person must inherit the defect from both parents.
The defect in the gene causes cystine to accumulate inside the kidney leading stones
containing the amino acid cystine.
• There is a defect in proximal renal tubular reabsorption of cystine therefore, urinary
excretion of cystine is increased. Being sparingly soluble, cystine deposits in the
kidneys and forms cystine stones. This same problem also affects ornithine, lysine,
and arginine, but only cystine is clinically significant as it is the only amino acid in
this group that will form stones.
• Cystathionine synthetase is severely deficient in homocystinuria. This impairs the
conversion of methionine into cysteine. Cysteine accumulates and is converted into
homocysteine. Urinary excretion of homocystine is increased.

Genetics of Cystinuria

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Symptoms include osteoporosis, dislocation of lenses in the eyes, mental

retardation, ischaemic vascular disease, blood in the urine, severe pain in the
side or the back, almost always on one side, nausea and vomiting, pain near
the groin, pelvis, or abdomen.
Treatment: Changes to diet, medications, and surgery are options for treating
the stones that form due to cystinuria. Dietary changes: the treatment consists
of a low-methionine, high-cysteine diet, pyridoxine supplements may be given
to activate the residual cystathionine synthetase, reduction in salt intake to less
than 2 grams per day has also been shown to be helpful in preventing stone
formation in human.

ALKAPTONURIA
• Alkaptonuria is an inborn error of tyrosine metabolism. It is due to
absence of homogentisate oxidase.
• Alkaptonuria is caused by a mutation on the homogentisate 1,2-
dioxygenase (HGD) gene. Homogentisate, an intermediate in
catabolism of tyrosine, cannot be metabolised and then is excreted
in urine. Freshly voided urine is normal in color, but urine becomes
dark on exposure to air due to oxidation of homogentisate by
oxygen.
• It also called black urine disease. This condition is rare, affecting 1
in 250,000 to 1 million people worldwide.

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DIAGNOSIS
• A urine test (urinalysis) is done to test for alkaptonuria.
• If ferric chloride is added to the urine, it will turn the urine a
black color in patients with this condition.
TREATMENT
• There’s no specific treatment for alkaptonuria. Low-protein diet
is recommended.
• Large doses of ascorbic acid, or vitamin C is given to slow down
the accumulation of homogentisic acid in the cartilage.
• Some patients benefit from high-dose vitamin C.

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Albinism
• Albinism (from Latin albus, "white“ also called achromia, achromasia, or
achromatosis) is a congenital disorder characterized by the complete or partial
absence of pigment in the skin, hair and eyes.
• This is due to absence or defect of tyrosinase, a copper-containing enzyme
involved in the production of melanin. Albinism results from inheritance of
recessive gene alleles and is known to affect all vertebrates, including humans.
• A person inherits one or more defective genes that cause them to be unable to
produce the normal amounts of a pigment called melanin. The genes are
located on "autosomal" chromosomes.
• Autosomes are the chromosomes that contain genes for general body
characteristics. Both parents must carry a defective gene to have a child with
albinism. When neither parent has albinism but both carry the defective gene,
there is a one in four chance that the baby will be born with albinism.

Treatment
• There is no real cure for albinism, but the goal of treatment is to relieve
symptoms.
• Treatment involves protecting the skin and eyes from the sun: Reduce
sunburn risk by avoiding the sun, using sunscreen, and covering up
completely with clothing when exposed to the sun.
• Sunscreen should have a high SPF. Sunglasses may relieve light
sensitivity.
• Glasses are often prescribed to correct vision problems and eye position.
• Eye muscle surgery is sometimes recommended to correct abnormal eye
movements.

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Characteristics: There is little to no melanin

(important pigment) in the eyes, skin, and hair.
Vision problems are a result of the low amounts of
melanin in albinos. The eyes are usually blue or
light brown, but can sometimes appear red. Skin
and hair is very pale in color. More likely to
sunburn and sensitivity to bright light
Types of Albinism
• Ocular Albinism (OA) affects only the eyes, not
the skin or hair. It results from an X-linked
chromosomal inheritance and so occurs mostly
in boys.
• Oculocutaneous Albinism (OCA): affects the
eyes, hair and skin and includes several different
forms. The first form, OCA1 involves the
tyrosinase enzyme, which converts tyrosine (an
amino acid) into melanin. Melanin is a chemical
that colors our skin, eyes and hair.

Tyrosinosis
• Since the first report of abnormal tyrosine metabolism by Medes in 19321 elevated blood
tyrosine has been reported in several different biochemical and phenotypical variations.
Tyrosinosis is an inherited inability of the body to metabolize tyrosine, para (p)
hydroxyphenylpyruvic acid to homogentisic acid because the enzyme
parahydroxyphenylpyruvic acid (PHPPA) oxidase is inactive.
• Disorders of tyrosine metabolism range from the benign condition of neonatal tyrosinemia to
a rapidly fatal hereditary tyrosinosis. The common biochemical denominator is increased
plasma tyrosine level and tyrosyluria (presence of tyrosine and its derivatives in urine). The
dysfunction in tyrosine degradation may be subdivided into four etiological groups:
• Group I: Tyrosine elevation secondary to severe liver damage, pernicious anemia or vitamin
C deficiency.
• Group II: Immaturity of the enzyme system, parahydroxyphenylpyruvic acid (PHPPA)
oxidase, as in neonatal tyrosinemia (benign hypertyrosinemia). This is a well-known
condition of transient elevation of serum tyrosine, together with tyrosyluria, occurring
occasionally in premature and in some term infants fed a high protein diet.
• Group III: Disorders described in the literature as tyrosinosis, hereditary tyrosinemia and
inborn hepatorenal dysfunction, in which tyrosinemia and tyrosyluria are associated with
liver or kidney damage.

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• Methioninemia, aminoaciduria and glycosuria have been almost constant

features of this form of tyrosinosis in the untreated state.
• In spite of early conjecture it has not been established that tyrosinosis associated
with hepatorenal disease is a primary defect of tyrosine metabolism and not a
consequence of liver disease. It is probable that decreased ability to metabolize
tyrosine and methionine is an independent secondary manifestation of a disease
process as yet unidentified. In its most frequently described form hereditary
tyrosinosis presents as an acute progressive illness starting in the neonatal
period.
• There are signs of hepatic failure, renal tubular dysfunction and vitamin D-
resistant rickets, and in 90% of cases death occurs in infancy. Children surviving
the first year of life show severe psychomotor retardation and most of them die
of hepatic and renal failure in the first decade.

• Group IV: More recently identified cases of tyrosinemia and tyrosyluria

without hepatorenal disease. These cases may actually be examples of
"essential tyrosinemia" . a primary genetic defect in tyrosine metabolism. For
lack of a better name this condition is usually referred to as "tyrosinosis
without hepatorenal disease".
• In the cases so far reported, this form of tyrosinosis is associated with mental
retardation. It runs a non-progressive course and, in spite of high plasma
tyrosine levels and tyrosyluria, there is no elevation of the methionine level
and both liver and kidney function remain normal.
• A dysfunction in tyrosine metabolism along the major pathway has been
identified in several patients having tyrosinosis with hepatorenal disease,
suggesting a block at the level of PHPPA-oxidase, and the deficient activity of
liver PHPPA oxidase has been measured.

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