Detoxification of ammonia and biosynthesis of urea.

Specific
pathways of amino acid metabolism. Mechanisms of hormonal
regulation and pathologies of protein metabolism.

Ammonia the highly toxic product of protein catabolism, is rapidly

inactivated by a variety of reactions. Some product of these reactions are utilized
for other purposes (thus salvaging a portion of the amino nitrogen), while others
are excreted. The excreted form varies quite widely among vertebrate and
invertebrate animals. The development of a pathway for nitrogen disposal in a
species appears to depend chiefly on the availability of water.

Humans are totally dependent on other organisms for converting

atmospheric nitrogen into forms available to the body. Nitrogen fixation is carried
out by bacterial nitrogenases forming reduced nitrogen, NH4+ which can then be
used by all organisms to form amino acids.
 Overview of the flow of nitrogen in the biosphere. Nitrogen, nitrites and
nitrates are acted upon by bacteria (nitrogen fixation) and plants and we assimilate
these compounds as protein in our diets. Ammonia incorporation in animals occurs
through the actions of glutamate dehydrogenase and glutamine synthase.
Glutamate plays the central role in mammalian nitrogen flow, serving as both a
nitrogen donor and nitrogen acceptor.

Reduced nitrogen enters the human body as dietary free amino acids,
protein, and the ammonia produced by intestinal tract bacteria. A pair of principal
enzymes, glutamate dehydrogenase and glutamine synthatase, are found in all
organisms and effect the conversion of ammonia into the amino acids glutamate
and glutamine, respectively. Amino and amide groups from these 2 substances are
freely transferred to other carbon skeletons by transamination and transamidation
reactions.

Aminotransferases exist for all amino acids except threonine and lysine. The
most common compounds involved as a donor/acceptor pair in transamination
reactions are glutamate and α-KG, which participate in reactions with many
different aminotransferases. Serum aminotransferases such as aspartate
aminotransferase, AST and alanine transaminase, ALT have been used as clinical
markers of tissue damage, with increasing serum levels indicating an increased
extent of damage. Alanine transaminase has an important function in the delivery
of skeletal muscle carbon and nitrogen (in the form of alanine) to the liver. In
skeletal muscle, pyruvate is transaminated to alanine, thus affording an additional
route of nitrogen transport from muscle to liver. In the liver, alanine transaminase
transfers the ammonia to α-KG and regenerates pyruvate. The pyruvate can then be
diverted into gluconeogenesis. This process is referred to as the glucose-alanine
cycle.
 The glucose-alanine cycle is used primarily as a mechanism for skeletal
muscle to eliminate nitrogen while replenishing its energy supply. Glucose
oxidation produces pyruvate which can undergo transamination to alanine. This
reaction is catalyzed by alanine transaminase, ALT. Additionally, during periods of
fasting, skeletal muscle protein is degraded for the energy value of the amino acid
carbons and alanine is a major amino acid in protein. The alanine then enters the
blood stream and is transported to the liver. Within the liver alanine is converted
back to pyruvate which is then a source of carbon atoms for gluconeogenesis. The
newly formed glucose can then enter the blood for delivery back to the muscle.
The amino group transported from the muscle to the liver in the form of alanine is
converted to urea in the urea cycle and excreted.

The reaction catalyzed by glutamate dehydrogenase is:

 The glutamate dehydrogenase utilizes both nicotinamide nucleotide
cofactors; NAD+ in the direction of nitrogen liberation and NADP+ for nitrogen
incorporation. In the forward reaction as shown above glutamate dehydrogenase is
important in converting free ammonia and α-KG to glutamate, forming one of the
20 amino acids required for protein synthesis. However, it should be recognized
that the reverse reaction is a key anapleurotic process linking amino acid
metabolism with TCA cycle activity. In the reverse reaction, glutamate
dehydrogenase provides an oxidizable carbon source used for the production of
energy as well as a reduced electron carrier, NADH. As expected for a branch
point enzyme with an important link to energy metabolism, glutamate
dehydrogenase is regulated by the cell energy charge. ATP and GTP are positive
allosteric effectors of the formation of glutamate, whereas ADP and GDP are
positive allosteric effectors of the reverse reaction. Thus, when the level of ATP is
high, conversion of glutamate to α-KG and other TCA cycle intermediates is
limited; when the cellular energy charge is low, glutamate is converted to ammonia
and oxidizable TCA cycle intermediates. Glutamate is also a principal amino donor
to other amino acids in subsequent transamination reactions. The multiple roles of
glutamate in nitrogen balance make it a gateway between free ammonia and the
amino groups of most amino acids.

The Glutamine Synthetase Reaction:

 The glutamine synthetase reaction is also important in several respects. First
it produces glutamine, one of the 20 major amino acids. Second, in animals,
glutamine is the major amino acid found in the circulatory system. Its role there is
to carry ammonia to and from various tissues but principally from peripheral
tissues to the kidney, where the amide nitrogen is hydrolyzed by the enzyme
glutaminase (reaction below); this process regenerates glutamate and free
ammonium ion, which is excreted in the urine.

Note that, in this function, ammonia arising in peripheral tissue is carried in

a non-ionizable form which has none of the neurotoxic or alkalosis-generating
properties of free ammonia.

Liver contains both glutamine synthetase and glutaminase but the enzymes
are localized in different cellular segments. This ensures that the liver is neither a
net producer nor consumer of glutamine. The differences in cellular location of
these two enzymes allows the liver to scavenge ammonia that has not been
incorporated into urea. The enzymes of the urea cycle are located in the same cells
as those that contain glutaminase. The result of the differential distribution of these
two hepatic enzymes makes it possible to control ammonia incorporation into
either urea or glutamine, the latter leads to excretion of ammonia by the kidney.

When acidosis occurs the body will divert more glutamine from the liver to
the kidney. This allows for the conservation of bicarbonate ion since the
incorporation of ammonia into urea requires bicarbonate (see below). When
glutamine enters the kidney, glutaminase releases one mole of ammonia generating
glutamate and then glutamate dehydrogenase releases another mole of ammonia
generating α-KG. The ammonia will ionizes to ammonium ion (NH4+) which is
excreted. The net effect is a reduction in the concentration of hydrogen ion, [H+],
and thus an increase in the pH.

THE UREA CYCLE

Earlier it was noted that kidney glutaminase was responsible for converting
excess glutamine from the liver to urine ammonium. However, about 80% of the
excreted nitrogen is in the form of urea which is also largely made in the liver, in a
series of reactions that are distributed between the mitochondrial matrix and the
cytosol. The series of reactions that form urea is known as the Urea Cycle or the
Krebs-Henseleit Cycle.
http://www.youtube.com/watch?v=AoBbVu5rnMs&feature=related

Enzymes:

1: carbamoyl phosphate synthetase-I (CPS-I)

2: ornithine transcarbamoylase (OTC)

3: argininosuccinate synthetase

4: argininosuccinase

5: arginase
 The essential features of the urea cycle reactions and their metabolic
regulation are as follows: arginine from the diet or from protein breakdown is
cleaved by the cytosolic enzyme arginase, generating urea and ornithine. In
subsequent reactions of the urea cycle a new urea residue is built on the ornithine,
regenerating arginine and perpetuating the cycle.

Ornithine arising in the cytosol is transported to the mitochondrial matrix,

where ornithine transcabamoylase catalyzes the condensation of ornithine with
carbamoyl phosphate, producing citrulline. The energy for the reaction is provided
by the high-energy anhydride of carbamoyl phosphate.

The product, citrulline, is then transported to the cytosol, where the

remaining reactions of the cycle take place. The synthesis of citrulline requires a
prior synthesis of carbamoyl phosphate (CP).
 The activation step requires 2 equivalents of ATP and the mitochondrial
matrix enzyme carbamoyl phosphate synthetase-I (CPS-I) (see reaction
mechanism).There are two CP synthetases: a mitochondrial enzyme, CPS-I, which
forms CP destined for inclusion in the urea cycle, and a cytosolic CP synthatase
(CPS-II), which is involved in pyrimidine nucleotide biosynthesis. CPS-I is
positively regulated by the allosteric effector N-acetyl-glutamate, while the
cytosolic enzyme is acetylglutamate independent.

In a 2-step reaction, catalyzed by cytosolic argininosuccinate synthetase,

citrulline is converted to argininosuccinate.
 The reaction involves the addition of AMP (from ATP) to the amido
carbonyl of citrulline, forming an activated intermediate on the enzyme surface
(AMP-citrulline), and the subsequent addition of aspartate to form
argininosuccinate.

Arginine and fumarate are produced from argininosuccinate by the cytosolic

enzyme argininosuccinate lyase.

In the final step of the cycle arginase cleaves urea from aspartate,
regenerating cytosolic ornithine, which can be transported to the mitochondrial
matrix for another round of urea synthesis.
 Beginning and ending with ornithine, the reactions of the cycle consumes 3
equivalents of ATP and a total of 4 high-energy nucleotide phosphates. Urea is the
only new compound generated by the cycle; all other intermediates and reactants
are recycled.

The energy consumed in the production of urea is more than recovered by

the release of energy formed during the synthesis of the urea cycle intermediates.
Ammonia released during the glutamate dehydrogenase reaction is coupled to the
formation of NADH. In addition, when fumarate is converted back to aspartate, the
malate dehydrogenase reaction used to convert malate to oxaloacetate generates a
mole of NADH. These two moles of NADH, thus, are oxidized in the mitochondria
yielding 6 moles of ATP.
 Regulation of the Urea Cycle

The urea cycle operates only to eliminate excess nitrogen. On high-protein

diets the carbon skeletons of the amino acids are oxidized for energy or stored as
fat and glycogen, but the amino nitrogen must be excreted. To facilitate this
process, enzymes of the urea cycle are controlled at the gene level. With long-term
changes in the quantity of dietary protein, changes of 20-fold or greater in the
concentration of cycle enzymes are observed. When dietary proteins increase
significantly, enzyme concentrations rise. On return to a balanced diet, enzyme
levels decline. Under conditions of starvation, enzyme levels rise as proteins are
degraded and amino acid carbon skeletons are used to provide energy, thus
increasing the quantity of nitrogen that must be excreted.

Short-term regulation of the cycle occurs principally at CPS-I, which is

relatively inactive in the absence of its allosteric activator N-acetylglutamate. The
steady-state concentration of N-acetylglutamate is set by the concentration of its
components acetyl-CoA and glutamate and by arginine, which is a positive
allosteric effector of N-acetylglutamate synthetase (glutamate transacylase).

Urea Cycle Defects (UCDs)

A complete lack of any one of the enzymes of the urea cycle will result in
death shortly after birth. However, deficiencies in each of the enzymes of the urea
cycle, including N-acetylglutamate synthase, have been identified. These disorders
are referred to as urea cycle disorders or UCDs. A common thread to most UCDs
is hyperammonemia leading to ammonia intoxication with the consequences
described below. Deficiencies in arginase do not lead to symptomatic
hyperammonemia as severe or as commonly as in the other UCDs.

Clinical symptoms are most severe when the UCD is at the level of
carbamoyl phosphate synthetase I. Symptoms of UCDs usually arise at birth and
encompass, ataxia, convulsions, lethargy, poor feeding and eventually coma and
death if not recognized and treated properly. In fact, the mortality rate is 100% for
UCDs that are left undiagnosed. Several UCDs manifest with late-onset such as in
adulthood. In these cases the symptoms are hyperactivity, hepatomegaly and an
avoidance of high protein foods.

In general, the treatment of UCDs has as common elements the reduction of

protein in the diet, removal of excess ammonia and replacement of intermediates
missing from the urea cycle. Administration of levulose reduces ammonia through
its action of acidifying the colon. Bacteria metabolize levulose to acidic byproducts
which then promotes excretion of ammonia in the feces as ammonium ions, NH 4+.
Antibiotics can be administered to kill intestinal ammonia producing bacteria.
Sodium benzoate and sodium phenylbutyrate can be administered to covalently
bind glycine (forming hippurate) and glutamine (forming phenylacetylglutamine),
respectively. These latter compounds, which contain the ammonia nitrogen, are
excreted in the feces. Dietary supplementation with arginine or citrulline can
increase the rate of urea production in certain UCDs.

Table of Urea Cycle Defects

UCD Enzyme Deficiency Symptoms/Comments

with 24h - 72h after birth infant

becomes lethargic, needs stimulation
to feed, vomiting, increasing lethargy,
hypothermia and hyperventilation;
Type I Carbamoylphosphate
without measurement of serum
Hyperammonemia synthetase I
ammonia levels and appropriate
intervention infant will die: treament
with arginine which activates N-
acetylglutamate synthetase

severe hyperammonemia, mild

hyperammonemia associated with
N-acetylglutamate deep coma, acidosis, recurrent
N-acetylglutamate
synthetase diarrhea, ataxia, hypoglycemia,
synthetase
Deficiency hyperornithinemia: treatment includes
administration of carbamoyl glutamate
to activate CPS I

Type 2 Ornithine most commonly occurring UCD, only

Hyperammonemia transcarbamoylase X-linked UCD, ammonia and amino
acids elevated in serum, increased
 serum orotic acid due to mitochondrial
carbamoylphosphate entering cytosol
and being incorporated into
pyrimidine nucleotides which leads to
excess production and consequently
excess catabolic products: treat with
high carbohydrate, low protein diet,
ammonia detoxification with sodium
phenylacetate or sodium benzoate

episodic hyperammonemia, vomiting,

lethargy, ataxia, siezures, eventual
Classic Argininosuccinate coma: treat with arginine
Citrullinemia synthetase administration to enhance citrulline
excretion, also with sodium benzoate
for ammonia detoxification

episodic symptoms similar to classic

Argininosuccinate citrullinemia, elevated plasma and
Argininosuccinic
lyase cerebral spinal fluid argininosuccinate:
aciduria
(argininosuccinase) treat with arginine and sodium
benzoate

rare UCD, progressive spastic

quadriplegia and mental retardation,
ammonia and arginine high in cerebral
Hyperargininemia Arginase spinal fluid and serum, arginine, lysine
and ornithine high in urine: treatment
includes diet of essential amino acids
excluding arginine, low protein diet

Protein obtained from the diet or from body protein during prolonged fasting or
starvation may be used as an energy source. Body protein is catabolized primarily
in muscle and in liver.

Amino acids released from proteins usually lose their amino group through
transamination or deamination. The carbon skeletons can be converted in the liver
to glucose (glucogenic amino acids), acetyl CoA, and ketone bodies (ketogenic),
or in a few cases both may be produced (glucogenic and ketogenic).

REMOVAL AND EXCRETION OF AMINO GROUPS

Excess nitrogen is eliminated from the body in the urine. The kidney adds small
quantities of ammonium ion to the urine in part to regulate acid-base balance, but
nitrogen is also eliminated in this process.

Most excess nitrogen is converted to urea in the liver and goes through the blood to
the kidney, where it is eliminated in urine. Amino groups released by deamination
reactions form ammonium ion (NH4+), which must not escape into the peripheral
blood. An elevated concentration of ammonium ion in the blood,
hyperammonemia, has toxic effects in the brain (cerebral edema, convulsions,
coma, and death).

Most tissues add excess nitrogen to the blood as glutamine by attaching ammonia
to the γ-carboxyl group of glutamate. Muscle sends nitrogen to the liver as alanine
and smaller quantities of other amino acids, in addition to glutamine. The figure
below summarizes the flow of nitrogen from tissues to the liver or kidney for
excretion.

Bridge to Pharmacology Lactulose is metabolized to lactic acid in the GI tract by

bacteria, which in turn covert ammonia (NH3) to ammonium (NH4+), interfering
with absorption and treating hyperammonemia.
 Figure I. Amino Group Removal for Elimination as Urea and Ammonia

Glutamine Synthetase Most tissues, including muscle, have glutamine synthetase,

which captures excess nitrogen by aminating glutamate to form glutamine. The
reaction is irreversible. Glutamine, a relatively nontoxic substance, is the major
carrier of excess nitrogen from tissues.

Glutaminase
The kidney contains glutaminase, allowing it to deaminate glutamine arriving in
the blood and to eliminate the amino group as ammonium ion in urine. The
reaction is irreversible. Kidney glutaminase is induced by chronic acidosis, in
which excretion of ammonium may become the major defense mechanism. The
liver has only small quantities of glutaminase; however, levels of the enzyme are
high in the intestine where the ammonium ion from deamination can be sent
directly to the liver via the portal blood and used for urea synthesis. The intestinal
bacteria and glutamine from dietary protein contribute to the intestinal ammonia
entering the portal blood.

UREA CYCLE Urea, which contains 2 nitrogens, is synthesized in the liver from
aspartate and carbamoyl phosphate, which in turn is produced from ammonium ion
and carbon dioxide by mitochondrial carbamoyl phosphate synthetase. This
enzyme requires N-acetylglutamate as an activator. N-acetylglutamate is produced
only when free amino acids are present.

NH4+ + HCO- + 2 ATP

Carbamoyl phosphate synthetase I

 Figure 2. The Urea Cycle in t lie Liver

The urea cycle, like the citric acid cycle, acts catalytically. Small quantities of the
intermediates are sufficient to synthesize large amounts of urea from aspartate and
carbamoyl phosphate.

The cycle occurs partially in the mitochondria and partially in the cytoplasm.

• Citrulline enters the cytoplasm, and ornithine returns to the mitochondria.

• Carbamoyl phosphate synthetase and ornithine transcarbamoylase are

mitochondrial enzymes.

• Aspartate enters the cycle in the cytoplasm and leaves the cycle (minus its amino
group) as fumarate. If gluconeogenesis is active, fumarate can be converted to
glucose.

• The product urea is formed in the cytoplasm and enters the blood for delivery to
the kidney
Genetic Deficiencies of the Urea Cycle

A combination of hyperammonemia, elevated blood glutamine, and decreased

blood urea nitrogen (BUN) suggests a defect in the urea cycle. With neonatal
onset, infants typically appear normal for the first 24 hours. Sometime during the
24- to 72-hour postnatal period, symptoms of lethargy, vomiting, and
hyperventilation begin and, if not treated, progress to coma, respiratory failure, and
death.

There are 2 deficiencies of the 2 mitochondrial enzymes in the urea cycle:

carbamoyl phosphate synthetase and ornithine transcarbamoylase.

They can be distinguished by an increase in orotic acid and uracil, which occurs in
ornithine transcarbamoylase deficiency, but not in the deficiency of carbamoyl
phosphate synthetase.

Orotic acid and uracil are intermediates in pyrimidine synthesis. This pathway is
stimulated by the accumulation of carbamoyl phosphate, the substrate for ornithine
transcarbamoylase in the urea cycle and for aspartate transcarbamoylase in
pyrimidine synthesis.

Table I. Genetic Deficiencies of Urea Synthesis

Carbamoyl Phosphate Synthetase Ornithine Transcarbamoylase

↑ [NH4+]; hyperammonemia ↑ [NH4+]; hyperammonemia

Blood glutamine is increased Blood glutamine is increased

BUN is decreased BUN is decreased

No orotic aciduria Autosomal Orotic aciduria X-linked recessive

recessive
Cerebral edema Cerebral edema

Lethargy, convulsions, coma, death Lethargy, convulsions, coma, death

These conditions can be treated with a low protein diet and administration of
sodium benzoate or phenylpyruvate to provide an alternative route for capturing
and excreting excess nitrogen.

Neurotoxicity Associated with Ammonia

Earlier it was noted that ammonia was neurotoxic. Marked brain damage is
seen in cases of failure to make urea via the urea cycle or to eliminate urea through
the kidneys. The result of either of these events is a buildup of circulating levels of
ammonium ion. Aside from its effect on blood pH, ammonia readily traverses the
brain blood barrier and in the brain is converted to glutamate via glutamate
dehydrogenase, depleting the brain of α-ketoglutarate. As the α-ketoglutarate is
depleted, oxaloacetate falls correspondingly, and ultimately TCA cycle activity
comes to a halt. In the absence of aerobic oxidative phosphorylation and TCA
cycle activity, irreparable cell damage and neural cell death ensue. In addition, the
increased glutamate leads to glutamine formation. This depletes glutamate stores
which are needed in neural tissue since glutamate is both a neurotransmitter and a
precursor for the synthesis of α -aminobutyrate, GABA, another neurotransmitter.
Therefore, reductions in brain glutamate affect energy production as well as
neurotransmission.

All tissues have some capability for synthesis of the non-essential amino
acids, amino acid remodeling, and conversion of non-amino acid carbon skeletons
into amino acids and other derivatives that contain nitrogen. However, the liver is
the major site of nitrogen metabolism in the body. In times of dietary surplus, the
potentially toxic nitrogen of amino acids is eliminated via transaminations,
deamination, and urea formation; the carbon skeletons are generally conserved as
carbohydrate, via gluconeogenesis, or as fatty acid via fatty acid synthesis
pathways. In this respect amino acids fall into three categories: glucogenic,
ketogenic, or glucogenic and ketogenic. Glucogenic amino acids are those that give
rise to a net production of pyruvate or TCA cycle intermediates, such as α-
ketoglutarate or oxaloacetate, all of which are precursors to glucose via
gluconeogenesis. All amino acids except lysine and leucine are at least partly
glucogenic. Lysine and leucine are the only amino acids that are solely ketogenic,
giving rise only to acetylCoA or acetoacetylCoA, neither of which can bring about
net glucose production.

A small group of amino acids comprised of isoleucine, phenylalanine,

threonine, tryptophan, and tyrosine give rise to both glucose and fatty acid
precursors and are thus characterized as being glucogenic and ketogenic. Finally, it
should be recognized that amino acids have a third possible fate. During times of
starvation the reduced carbon skeleton is used for energy production, with the
result that it is oxidized to CO2 and H2O.

AMINO ACID BIOSYNTHESIS

Glutamate and Aspartate

Glutamate and aspartate are synthesized from their widely distributed α-keto
acid precursors by simple transamination reactions. The former catalyzed by
glutamate dehydrogenase and the latter by aspartate aminotransferase, AST.
Aspartate is also derived from asparagine through the action of asparaginase. The
importance of glutamate as a common intracellular amino donor for transamination
reactions and of aspartate as a precursor of ornithine for the urea cycle.
 Alanine and the Glucose-Alanine Cycle

Aside from its role in protein synthesis, alanine is second only to glutamine
in prominence as a circulating amino acid. In this capacity it serves a unique role in
the transfer of nitrogen from peripheral tissue to the liver. Alanine is transferred to
the circulation by many tissues, but mainly by muscle, in which alanine is formed
from pyruvate at a rate proportional to intracellular pyruvate levels. Liver
accumulates plasma alanine, reverses the transamination that occurs in muscle, and
proportionately increases urea production. The pyruvate is either oxidized or
converted to glucose via gluconeogenesis. When alanine transfer from muscle to
liver is coupled with glucose transport from liver back to muscle, the process is
known as the glucose-alanine cycle. The key feature of the cycle is that in 1
molecule, alanine, peripheral tissue exports pyruvate and ammonia (which are
potentially rate-limiting for metabolism) to the liver, where the carbon skeleton is
recycled and most nitrogen eliminated. There are 2 main pathways to production of
muscle alanine: directly from protein degradation, and via the transamination of
pyruvate by glutamate-pyruvate aminotransferase.

Cysteine Biosynthesis

The sulfur for cysteine synthesis comes from the essential amino acid
methionine. A condensation of ATP and methionine catalyzed by methionine
adenosyltransferase S-adenosylmethionine (SAM or AdoMet).
 Biosynthesis of S-adenosylmethionine, SAM

SAM serves as a precurosor for numerous methyl transfer reactions the

conversion of norepinephrine to epinenephrine. The result of methyl transfer is the
conversion of SAM to S-adenosylhomocysteine. S-adenosylhomocysteine is then
cleaved by adenosylhomocyteinase to yield homocysteine and adenosine.
Homocysteine can be converted back to methionine by methionine synthase, a
reaction that occurs under methionine-sparing conditions and requires N5-methyl-
tetrahydrofolate as methyl donor. This reaction was discussed in the context of
vitamin B12-requiring enzymes. Transmethylation reactions employing SAM are
extremely important, but in this case the role of S-adenosylmethionine in
transmethylation is secondary to the production of homocysteine (essentially a by-
product of transmethylase activity). In the production of SAM all phosphates of an
ATP are lost: one as Pi and two as PPi. It is adenosine which is transferred to
methionine and not AMP. In cysteine synthesis, homocysteine condenses with
serine to produce cystathionine, which is subsequently cleaved by cystathionase to
produce cysteine and α-ketobutyrate. The sum of the latter two reactions is known
as trans-sulfuration. Cysteine is used for protein synthesis and other body needs,
while the α- ketobutyrate is decarboxylated and converted to propionyl-CoA.
While cysteine readily oxidizes in air to form the disulfide cystine, cells contain
little if any free cystine because the ubiquitous reducing agent, glutathione
effectively reverses the formation of cystine by a non-enzymatic reduction
reaction.
 Utilization of methionine in the synthesis of cysteine

The 2 key enzymes of this pathway, cystathionine synthase and

cystathionase (cystathionine lyase), both use pyridoxal phosphate as a cofactor,
and both are under regulatory control. Cystathionase is under negative allosteric
control by cysteine, as well, cysteine inhibits the expression of the cystathionine
synthase gene. Genetic defects are known for both the synthase and the lyase.
Missing or impaired cystathionine synthase leads to homocystinuria and is often
associated with mental retardation, although the complete syndrome is
multifaceted and many individuals with this disease are mentally normal. Some
instances of genetic homocystinuria respond favorably to pyridoxine therapy,
suggesting that in these cases the defect in cystathionine synthase is a decreased
affinity for the cofactor. Missing or impaired cystathionase leads to excretion of
cystathionine in the urine but does not have any other untoward effects. Rare cases
are known in which cystathionase is defective and operates at a low level. This
genetic disease leads to methioninuria with no other consequences.

Tyrosine Biosynthesis

Tyrosine is produced in cells by hydroxylating the essential amino acid

phenylalanine. This relationship is much like that between cysteine and
methionine. Half of the phenylalanine required goes into the production of
tyrosine; if the diet is rich in tyrosine itself, the requirements for phenylalanine are
reduced by about 50%. Phenylalanine hydroxylase is a mixed-function oxygenase:
one atom of oxygen is incorporated into water and the other into the hydroxyl of
tyrosine. The reductant is the tetrahydrofolate-related cofactor tetrahydrobiopterin,
which is maintained in the reduced state by the NADH-dependent enzyme
dihydropteridine reductase.
 Biosynthesis of tyrosine from phenylalanine

Missing or deficient phenylalanine hydroxylase leads to the genetic disease

known as phenlyketonuria (PKU), which if untreated leads to severe mental
retardation. The mental retardation is caused by the accumulation of phenylalanine,
which becomes a major donor of amino groups in aminotransferase activity and
depletes neural tissue of α-ketoglutarate. This absence of α-ketoglutarate in the
brain shuts down the TCA cycle and the associated production of aerobic energy,
which is essential to normal brain development.

The product of phenylalanine transamination, phenylpyruvic acid, is reduced

to phenylacetate and phenyllactate, and all 3 compounds appear in the urine. The
presence of phenylacetate in the urine imparts a "mousy" odor. If the problem is
diagnosed early, the addition of tyrosine and restriction of phenylalanine from the
diet can minimize the extent of mental retardation.

In other pathways, tetrahydrobiopterin is a cofactor. The effects of missing

or defective dihydropteridine reductase cause even more severe neurological
difficulties than those usually associated with PKU caused by deficient
hydroxylase activity.

Ornithine and Proline Biosynthesis

Glutamate is the precursor of both proline and ornithine, with glutamate

semialdehyde being a branch point intermediate leading to one or the other of these
2 products. While ornithine is not one of the 20 amino acids used in protein
synthesis, it plays a significant role as the acceptor of carbamoyl phosphate in the
urea cycle. Ornithine serves an additional important role as the precursor for the
synthesis of the polyamines. The production of ornithine from glutamate is
important when dietary arginine, the other principal source of ornithine, is limited.
The fate of glutamate semialdehyde depends on prevailing cellular conditions.
Ornithine production occurs from the semialdehyde via a simple glutamate-
dependent transamination, producing ornithine.

Ornithine synthesis from glutamate

When arginine concentrations become elevated, the ornithine contributed

from the urea cycle plus that from glutamate semialdehyde inhibit the
aminotransferase reaction, with accumulation of the semialdehyde as a result. The
semialdehyde cyclizes spontaneously to pyrroline-5-carboxylate which is then
reduced to proline by an NADPH-dependent reductase.

Serine Biosynthesis

The main pathway to serine starts with the glycolytic intermediate 3-

phosphoglycerate.

An NADH-linked dehydrogenase converts 3-phosphoglycerate into a keto

acid, 3-phosphopyruvate, suitable for subsequent transamination. Aminotransferase
activity with glutamate as a donor produces 3-phosphoserine, which is converted to
serine by phosphoserine phosphatase.
 Glycine Biosynthesis

The main pathway to glycine is a 1-step reaction catalyzed by serine

hydroxymethyltransferase.

This reaction involves the transfer of the hydroxymethyl group from serine
to the cofactor tetrahydrofolate (THF), producing glycine and N5,N10-methylene-
THF. Glycine produced from serine or from the diet can also be oxidized by
glycine cleavage complex, GCC, to yield a second equivalent of N5,N10-
methylene-tetrahydrofolate as well as ammonia and CO2.
 Glycine is involved in many anabolic reactions other than protein synthesis
including the synthesis of purine nucleotides, heme, glutathione, creatine and
serine.

Aspartate/Asparagine and Glutamate/Glutamine Biosynthesis

Glutamate is synthesized by the reductive amination of α-ketoglutarate

catalyzed by glutamate dehydrogenase; it is thus a nitrogen-fixing reaction. In
addition, glutamate arises by aminotransferase reactions, with the amino nitrogen
being donated by a number of different amino acids. Thus, glutamate is a general
collector of amino nitrogen. Aspartate is formed in a transamintion reaction
catalyzed by aspartate transaminase, AST. This reaction uses the aspartate α-keto
acid analog, oxaloacetate, and glutamate as the amino donor. Aspartate can also be
formed by deamination of asparagine catalyzed by asparaginase. Asparagine
synthetase and glutamine synthetase, catalyze the production of asparagine and
glutamine from their respective α- amino acids. Glutamine is produced from
glutamate by the direct incorporation of ammonia; and this can be considered
another nitrogen fixing reaction. Asparagine, however, is formed by an
amidotransferase reaction. Aminotransferase reactions are readily reversible. The
direction of any individual transamination depends principally on the concentration
ratio of reactants and products. By contrast, transamidation reactions, which are
dependent on ATP, are considered irreversible. As a consequence, the degradation
of asparagine and glutamine take place by a hydrolytic pathway rather than by a
reversal of the pathway by which they were formed. As indicated above,
asparagine can be degraded to aspartate.

PATHWAYS OF AMINO ACID DEGRADATION

There are 20 standard amino acids in proteins, with a variety of carbon

skeletons. Correspondingly, there are 20 different catabolic pathways for amino
acid degradation. In humans, these pathways taken together normally account for
only 10 to 15% of the body's energy production. Therefore, the individual amino
acid degradative pathways are not nearly as active as glycolysis and fatty acid
oxidation. In addition, the activity of the catabolic pathways can vary greatly from
one amino acid to the next, depending upon the balance between requirements for
biosynthetic processes and the amounts of a given amino acid available. For this
reason, we shall not examine them all in detail. The 20 catabolic pathways
converge to form only five products, all of which enter the citric acid cycle. From
here the carbons can be diverted to gluconeogenesis or ketogenesis, or they can be
completely oxidized to CO2 and H2O.
 All or part of the carbon skeletons of ten of the amino acids are ultimately
broken down to yield acetyl-CoA. Five amino acids are converted into α-
ketoglutarate, four into succinyl-CoA, two into fumarate, and two into
oxaloacetate. The individual pathways for the 20 amino acids will be summarized
by means of flow diagrams, each leading to a specific point of entry into the citric
acid cycle. In these diagrams the amino acid carbon atoms that enter the citric acid
cycle are shown in color. Note that some amino acids appear more than once,
reflecting the fact that different parts of their carbon skeletons have different fates.
Some of the enzymatic reactions in these pathways that are particularly noteworthy
for their mechanisms or their medical significance will be singled out for special
discussion.

Several Enzyme Cofactors Play Important Roles in Amino Acid

Catabolism

A variety of interesting chemical rearrangements are found among the amino

acid catabolic pathways. Before examining the pathways themselves, it is useful to
note classes of reactions that recur and to introduce the enzymatic cofactors
required. We have already considered one important class, the transamination
reactions requiring pyridoxal phosphate. Another common type of reaction in
amino acid catabolism is a one-carbon transfer. One-carbon transfers usually
involve one of three cofactors: biotin, tetrahydrofolate, or S-adenosylmethionine.

These cofactors are used to transfer one-carbon groups in different oxidation

states. The most oxidized state of carbon, CO2, is transferred by biotin. The
remaining two cofactors are especially important in amino acid and nucleotide
metabolism.

Tetrahydrofolate is generally involved in transfers of one-carbon groups in

the intermediate oxidation states, and S-adenosylmethionine in transfers of methyl
groups, the most reduced state of carbon.
 Tetrahydrofolate (H4 folate) consists of a substituted pteridine, p-
aminobenzoate, and glutamate linked together. This cofactor is synthesized in
bacteria and its precursor, folate, is a vitamin for mammals. The one-carbon group,
in any of three oxidation states, is bonded to N-5 or N-10 or to both. The most
reduced form of the cofactor carries a methyl group, a more oxidized form carries a
methylene group, and the most oxidized forms carry a methenyl, formyl, or
formimino group. The different forms of tetrahydrofolate are interconvertible and
serve as donors of one-carbon units in a variety of biosynthetic reactions. The
major source of one-carbon units for tetrahydrofolate is the carbon removed in the
conversion of serine to glycine, producing N5,N10-methylenetetrahydrofolate.

Although tetrahydrofolate can carry a methyl group at N-5, the methyl

group's transfer potential is insufficient for most biosynthetic reactions. S-
Adenosylmethionine is more commonly used for methyl group transfers. It is
synthesized from ATP and methionine by the action of methionine adenosyl
transferase. This reaction is unusual in that the nucleophilic sulfur atom of
methionine attacks at the 5' carbon of the ribose moiety of ATP, releasing
triphosphate, rather than attacking at one of the phosphorus atoms. The
triphosphate is cleaved to Pi and PPi on the enzyme, and the PPi is later cleaved by
inorganic pyrophosphatase, so that three bonds, two of which are high-energy
bonds, are broken in this reaction. The only other reaction known in which
triphosphate is displaced from ATP occurs in the synthesis of coenzyme B12.

S-Adenosylmethionine is a potent alkylating agent by virtue of its

destabilizing sulfonium ion. The methyl group is subject to attack by nucleophiles
and is about 1,000 times more reactive than the methyl group of N5-
methyltetrahydrofolate.

Transfer of a methyl group from S-adenosylmethionine to an acceptor yields

S-adenosylhomocysteine, which is subsequently broken down to homocysteine and
adenosine. Methionine is regenerated by the transfer of a methyl group to
homocysteine in a reaction catalyzed by methionine synthase. One form of this
enzyme is common in bacteria and uses N5-methyltetrahydrofolate as a methyl
donor. Another form that occurs in bacteria and mammals uses methylcobalamin
derived from coenzyme B12. This reaction and the rearrangement of L-
methylmalonyl-CoA to succinyl-CoA are the only coenzyme Bl2-dependent
reactions known in mammals. Methionine is reconverted to S-adenosylmethionine
to complete an activated methyl cycle.

Tetrahydrobiopterin is another cofactor introduced in these pathways, but it

is not involved in one-carbon transfers. Tetrahydrobiopterin is structurally related
to the flavin coenzymes, and it participates in biological oxidation reactions. It
belongs to a widespread class of biological compounds called pterins, and we will
consider its mode of action when we discuss phenylalanine degradation.

The carbon skeletons of ten amino acids yield acetyl-CoA, which enters the
citric acid cycle directly. Five of the ten are degraded to acetyl-CoA via pyruvate.
The other five are converted into acetyl-CoA and/or acetoacetyl-CoA, which is
then cleaved to form acetyl-CoA.

The five amino acids entering via pyruvate are alanine, glycine, serine,
cysteine, and tryptophan. In some organisms threonine is also degraded to form
acetyl-CoA, in humans it is degraded to succinyl-CoA, as described later. Alanine
yields pyruvate directly on transamination with a-ketoglutarate, and the side chain
of tryptophan is cleaved to yield alanine and thus pyruvate. Cysteine is converted
to pyruvate in two steps, one to remove the sulfur atom, the other a transamination.
Serine is converted to pyruvate by serine dehydratase. Both the β-hydroxyl and the
α-amino groups of serine are removed in this single PLP-dependent reaction.
Glycine has two pathways. It can be converted into serine by enzymatic addition of
a hydroxymethyl group. This reaction, catalyzed by serine hydroxymethyl
transferase, requires the coenzymes tetrahydrofolate and pyridoxal phosphate. The
second pathway for glycine, which predominates in animals, involves its oxidative
cleavage into CO2, NH4+ , and a methylene group (-CH2-). This readily reversible
reaction, catalyzed by glycine synthase, also requires tetrahydrofolate, which
accepts the methylene group. In this oxidative cleavage pathway the two carbon
atoms of glycine do not enter the citric acid cycle. One is lost as CO 2, and the other
becomes the methylene group of N5,N10- methylene- tetrahydrofolate, which is
used as a one-carbon group donor in certain biosynthetic pathways.

Portions of the carbon skeleton of six amino acids-tryptophan, lysine,

phenylalanine, tyrosine, leucine, and isoleucine-yield acetyl-CoA and/or
acetoacetyl-CoA; the latter is then converted into acetyl-CoA. Some of the final
steps in the degradative pathways for leucine, lysine, and tryptophan resemble
steps in the oxidation of fatty acids. The breakdown of two of these six amino
acids deserves special mention.
 Arginine Catabolism

The catabolism of arginine begins within the context of the urea cycle. It is
hydrolyzed to urea and ornithine by arginase.

The Arginine Metabolic Pathways: Nitric Oxide Synthase and Arginase

 Two important metabolic pathways use the amino acid arginine as the
precursor: the enzyme nitric oxide synthase, which converts arginine to nitric
oxide, and citrulline and the enzyme arginase, which converts arginine to ornithine
and urea. The latter is part of a pathway for detoxifying ammonia. Ornithine is also
part of a proliferative pathway that is involved in cell division and tissue
regeneration. Arginase II is the form of arginase that is thought to be involved in
the synthesis of polyamines, which control cell proliferation and collagen
production. It is most highly expressed in the prostate and kidney.1

There has been considerable recent publication of papers on nitric oxide

synthase because of the importance of nitric oxide in functions such as
(importantly) vasodilation (endothelial function). Scientists have found that an
inadequate supply of arginine or too little of the cofactor tetrahydrobiopterin
(which one paper reports may be mimicked by folic acid2) results in an
“uncoupling” of nitric oxide synthase from the production of nitric oxide,
producing superoxide anion instead. Not only is there a reduction in the production
of nitric oxide when nitric oxide synthase is uncoupled, but oxidative stress is
greatly increased.

Now, another major mechanism of decreased production of nitric oxide has

been reported: an increase in the arginase pathway for the use of arginine. Recent
studies have reported increases in arginase in conditions including reperfusion
injury, asthma, psoriasis, arthritis, and human breast cancer. (Since arginase II is
highly expressed in the prostate, it would be interesting to see whether there is
increased expression in prostate cancer.) The increased arginase decreases arginine
availability to be converted to nitric oxide, as well as increasing ornithine that can
be converted into polyamines, procellular proliferation factors. In psoriasis, for
example, there is hyperproliferation of keratinocytes. In the arthritis paper, it was
reported that arginase II could be induced ex vivo (outside the body) by
inflammatory factors such as PGE2 and LPS (lipopolysaccharide, from bacteria).
Ornithine, produced by arginase, is necessary for the production of collagen, which
occurs in rheumatoid arthritis.

Ornithine, in excess of urea cycle needs, is transaminated to form glutamate

semialdehyde.

Nitric Oxide Synthases (NOS)

Is an important cellular-signaling molecule, a potent vasodilatator due to the

smooth muscle relaxation. It also inhibits platelet adherence and aggregation,
reduces adherence of leukocytes to the endothelium. Furthermore, NO has been
shown to inhibit DNA synthesis and mitogenesis, and the proliferation of vascular
smooth muscle cells. These antiproliferative effects are likely to be mediated by
cyclic GMP.

Nitric Oxide Synthases from the biochemical point of view, are a family of
complex enzymes catalyzing the oxidation of L-arginine to form NO and L-
citrulline. The three human NOS isoforms identified to date are: eNOS (endothelial
NOS), nNOS (neuronal NOS), and iNOS (inducible NOS). Their genes are found
on human chromosomes 7, 12, and 17, respectively, and so they were named for
the tissue in which they were first cloned and characterized. vasculoprotective
effect of individual NOS isoforms in human organism is not sufficiently clarified
yet. Endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively
expressed, mainly in endothelial cells and nitrergic nerves, respectively,
synthesizing a small amount of NO under basal conditions and on stimulation by
various agonists. By contrast, inducible NOS (iNOS) is expressed when stimulated
by inflammatory stimuli, synthesizing a large amount of NO in a transient manner.
The knowledge of nitric oxide synthases (NOSs) is of extreme scientific
importance, not only for understanding new pathophysiological mechanisms but
also as a target for therapeutic intervention.
 The role of NO in regulating vascular tone and mediating platelet function is
attributable to the ongoing activity of eNOS. It is pharmacologically identical with
previously isolated EDRF (endothelium-derived releasing factor), exprimed by the
intact endothelium. Inactivation of the eNOS pathway limits the contribution of
NO to vessel homeostasis and results in increased vascular tone and platelet
adhesion and aggregation. The activity of eNOS is regulated by the intracellular
free calcium concentration and calcium- calmodulin complexes. Endothelial NOS
is a constitutively expressed protein predominantly associated with the particulate
subcellular fraction, suggesting that the native enzyme is a membrane-bound
protein. A detailed analysis of the membrane association of eNOS showed that this
enzyme is localized to the Golgi apparatus as well as to specific structures in the
plasmalemmal membrane called caveolae. The association of eNOS with a region
of the plasma membrane in which several key signal-transducing complexes are
concentrated (such as G-proteins) is likely to have profound repercussions on
enzyme activity as well as on its accessibility to intracellular mechanisms of the
pathway release, including mechanisms independent of intracellular calcium
release.

Neuronal Constitutive Nitric Oxide Synthase (nNOS) is present in central

and peripheral neuronal cells and certain epithelial cells. Its activity is also
regulated by Ca2+ and calmodulin. Its functions include long-term regulation of
synaptic transmission in the central nervous system, central regulation of blood
pressure, smooth muscle relaxation, and vasodilation via peripheral nitrergic
nerves. It has also been implicated in neuronal death in cerebrovascular stroke. NO
plays also an important role in the pathophysiology of some neurodegenerative
diseases. The presence of NO and NOS should be proved indirectly through the
histochemic positivity of nicotinamide dinucleotide phosphate diaphorase
(NADPHd). It was proposed that nerve stimulation directly activated the release of
NO from nitrergic nerves and, in fact, NO appears to be the dominant
neurotransmitter responsible for the nerve-mediated, endothelium-independent
vasodilation.
 Glycine Catabolism

Glycine is classified as a glucogenic amino acid, since it can be converted to

serine by serine hydroxymethyltransferase, and serine can be converted back to the
glycolytic intermediate, 3-phosphoglycerate or to pyruvate by serine/threonine
dehydratase.

Nevertheless, the main glycine catabolic pathway leads to the production of

CO2, ammonia, and one equivalent of N5,N10-methyleneTHF by the
mitochondrial glycine cleavage complex.
 Hyperglycinemia refers to a condition where glycine is elevated in the blood.

Types include:

- Propionic acidemia, also known as "ketotic glycinemia"

- Glycine encephalopathy, also known as "non-ketotic hyperglycinemia".
Glycine encephalopathy (also known as non-ketotic hyperglycinemia or
NKH) is a rare autosomal recessive disorder of glycine metabolism. After
phenylketonuria, glycine encephalopathy is the second most common disorder of
amino acid metabolism. The disease is caused by defects in the glycine cleavage
system, an enzyme responsible for glycine catabolism. There are several forms of
the disease, with varying severity of symptoms and time of onset. The symptoms
are exclusively neurological in nature, and clinically this disorder is characterized
by abnormally high levels of the amino acid glycine in bodily fluids and tissues,
especially the cerebral spinal fluid.

Glycine encephalopathy is sometimes referred to as "nonketotic

hyperglycinemia" (NKH), as a reference to the biochemical findings seen in
patients with the disorder, and to distinguish it from the disorders that cause
"ketotic hyperglycinemia" (seen in propionic acidemia and several other inherited
metabolic disorders). To avoid confusion, the term "glycine encephalopathy" is
often used, as this term more accurately describes the clinical symptoms of the
disorder. Glycine is metabolized in the body to end products of carbon dioxide and
ammonia. The glycine cleavage system, which is responsible for glycine
metabolism in the mitochondrion is made up of four protein subunits.

Propionic acidemia, also known as propionic aciduria, propionyl-CoA

carboxylase deficiency and ketotic glycinemia, is an autosomal recessive metabolic
disorder, classified as a branched-chain organic acidemia.

The disorder presents in the early neonatal period with progressive

encephalopathy. Death can occur quickly, due to secondary hyperammonemia,
infection, cardiomyopathy, or basal ganglial stroke.

Propionic Acidemia is a rare disorder that is inherited from both parents.

Being autosomal recessive, neither parent shows symptoms, but both carry a
defective gene responsible for this disease. It takes two faulty genes to cause PA,
so there is a 1 in 4 chance for these parents to have a child with PA. Propionic
acidemia is characterized almost immediately in newborns. Symptoms include
poor feeding, vomiting, dehydration, acidosis, low muscle tone (hypotonia),
seizures, and lethargy. The effects of propionic acidemia quickly become life-
threatening.

Glutamine/Glutamate and Asparagine/Aspartate Catabolism

Glutaminase is an important kidney tubule enzyme involved in converting

glutamine (from liver and from other tissue) to glutamate and NH3+, with the
NH3+ being excreted in the urine. Glutaminase activity is present in many other
tissues as well, although its activity is not nearly as prominent as in the kidney. The
glutamate produced from glutamine is converted to -ketoglutarate, making
glutamine a glucogenic amino acid. Asparaginase is also widely distributed within
the body, where it converts asparagine into ammonia and aspartate. Aspartate
transaminates to oxaloacetate, which follows the gluconeogenic pathway to
glucose. Glutamate and aspartate are important in collecting and eliminating amino
nitrogen via glutamine synthetase and the urea cycle, respectively. The catabolic
path of the carbon skeletons involves simple 1-step aminotransferase reactions that
directly produce net quantities of a TCA cycle intermediate. The glutamate
dehydrogenase reaction operating in the direction of -ketoglutarate production
provides a second avenue leading from glutamate to gluconeogenesis.

Alanine Catabolism

Alanine is also important in intertissue nitrogen transport as part of the

glucose-alanine cycle. Alanine's catabolic pathway involves a simple
aminotransferase reaction that directly produces pyruvate. Generally pyruvate
produced by this pathway will result in the formation of oxaloacetate, although
when the energy charge of a cell is low the pyruvate will be oxidized to CO2 and
H2O via the PDH complex and the TCA cycle. This makes alanine a glucogenic
amino acid.

Serine Catabolism

The conversion of serine to glycine and then glycine oxidation to CO2 and
NH3, with the production of two equivalents of N5,N10-methyleneTHF, was
described above. Serine can be catabolized back to the glycolytic intermediate, 3-
phosphoglycerate, by a pathway that is essentially a reversal of serine biosynthesis.
However, the enzymes are different. Serine can also be converted to pyruvate
through a deamination reaction catalyzed by serine/threonine dehydratase.

Proline Catabolism

Glutamine is converted to glutamate by glutaminase or several other

enzymes by the removal of the amide nitrogen. Proline is first converted to a Schiff
base and then converted by hydrolysis to glutamate-5-semialdehyde. All of these
changes occur on the same carbon. Arginine and histidine contain 5adjacent
carbons and a sixth carbon attached through a nitrogen attom. The catabolism of
these amino acids is thus slightly more complicated than glutamine or proline.
Arginine is converted to ornithine and urea. Ornithine is furthere transaminated to
produce glutamate-5-semialdehyde. Glutamate-5-semialdehyde is converted to
glutamate. The enzymes involved in the steps of the histidine pathway are listed in
the box in the lower right corner of the diagram. Tetrahydrofolate is the cofactor in
the final step converting histidine to glutamate. Transamination or deamination of
glutamate produces a-ketoglutarate which feeds into the citric acid cycle.

Glutamate semialdehyde can serve as the precursor for proline biosynthesis

as described above or it can be converted to glutamate. Proline catabolism is a
reversal of its synthesis process. The glutamate semialdehyde generated from
ornithine and proline catabolism is oxidized to glutamate by an ATP-independent
glutamate semialdehyde dehydrogenase. The glutamate can then be converted to
ketoglutarate in a transamination reaction. Thus arginine, ornithine and proline,
are glucogenic.
 Hyperprolinemia is an excess of a particular protein building block (amino
acid), called proline, in the blood. This condition generally occurs when proline is
not broken down properly by the body. There are two inherited forms of
hyperprolinemia, called type I and type II.

People with hyperprolinemia type I often do not show any symptoms,

although they have proline levels in their blood between 3 and 10 times the normal
level. Some individuals with hyperprolinemia type I exhibit seizures, intellectual
disability, or other neurological or psychiatric problems.

Hyperprolinemia type II results in proline levels in the blood between 10 and

15 times higher than normal, and high levels of a related compound called
pyrroline-5-carboxylate. This form of the disorder has signs and symptoms that
vary in severity, and is more likely than type I to involve seizures or intellectual
disability.
 Hyperprolinemia can also occur with other conditions, such as malnutrition
or liver disease. In particular, individuals with conditions that cause elevated levels
of lactic acid in the blood (lactic acidemia) may have hyperprolinemia as well,
because lactic acid inhibits the breakdown of proline.

Mutations in the ALDH4A1 and PRODH genes cause hyperprolinemia.

Inherited hyperprolinemia is caused by deficiencies in the enzymes that

break down (degrade) proline. Hyperprolinemia type I is caused by a mutation in
the PRODH gene, which provides instructions for producing the enzyme proline
oxidase. This enzyme begins the process of degrading proline by starting the
reaction that converts it to pyrroline-5-carboxylate.

Hyperprolinemia type II is caused by a mutation in the ALDH4A1 gene,

which provides instructions for producing the enzyme pyrroline-5-carboxylate
dehydrogenase. This enzyme helps to break down the pyrroline-5-carboxylate
produced in the previous reaction, converting it to the amino acid glutamate. The
conversion between proline and glutamate, and the reverse reaction controlled by
different enzymes, are important in maintaining a supply of the amino acids needed
for protein production, and for energy transfer within the cell.

A deficiency of either proline oxidase or pyrroline-5-carboxylate

dehydrogenase results in a buildup of proline in the body. A deficiency of the latter
enzyme leads to higher levels of proline and a buildup of the intermediate
breakdown product pyrroline-5-carboxylate, causing the signs and symptoms of
hyperprolinemia type II.

Histidine Catabolism

Histidine catabolism begins with release of the α- amino group catalyzed

by histidase, introducing a double bond into the molecule. As a result, the
deaminated product, urocanate, is not the usual α-keto acid associated with loss of
α-amino nitrogens. The end product of histidine catabolism is glutamate, making
histidine one of the glucogenic amino acids. Another key feature of histidine
catabolism is that it serves as a source of ring nitrogen to combine with
tetrahydrofolate (THF), producing the carbon THF intermediate known as N5-
formiminoTHF. The latter reaction is one of two routes to N5-formiminoTHF. The
principal genetic deficiency associated with histidine metabolism is absence or
deficiency of the first enzyme of the pathway, histidase.

The resultant histidinemia is relatively benign. The disease, which is of

relatively high incidence (1 in 10,000), is most easily detected by the absence of
urocanate from skin and sweat, where it is normally found in relative abundance.
Decarboxylation of histidine in the intestine by bacteria gives rise to histamine.
Similarly, histamine arises in many tissues by the decarboxylation of histidine,
which in excess causes constriction or dilation of various blood vessels. The
general symptoms are those of asthma and various allergic reactions.

Histamine, biologically active substance found in a great variety of living

organisms. It is distributed widely, albeit unevenly, throughout the animal
kingdom and is present in many plants and bacteria and in insect venom.
Histamine is chemically classified as an amine, an organic molecule based on the
structure of ammonia (NH3). It is formed by the decarboxylation (the removal of a
carboxyl group) of the amino acid histidine.

Histamine is synthesized in all tissues, but is particularly abundant in skin,

lung and gastrointestinal tract. Mast cells, which are present in many tissues, are a
prominent source of histamine, but histamine is also secreted by a number of other
immune cells. Mast cells have surface receptors that bind immunoglobulin E, and
when antigen crosslinks IgE on the mast cell surface, they respond by secreting
histamine, along with a variety of other bioactive mediators.
 Valine, Leucine and Isoleucine Catabolism

This group of essential amino acids are identified as the branched-chain

amino acids, BCAAs. Because this arrangement of carbon atoms cannot be made
by humans, these amino acids are an essential element in the diet. The catabolism
of all three compounds initiates in muscle and yields NADH and FADH2 which
can be utilized for ATP generation. The catabolism of all three of these amino
acids uses the same enzymes in the first two steps. The first step in each case is a
transamination using a single BCAA aminotransferase, with α-ketoglutarate as
amine acceptor. As a result, three different α-keto acids are produced and are
oxidized using a common branched-chain α-keto acid dehydrogenase, yielding the
three different CoA derivatives. Subsequently the metabolic pathways diverge,
producing many intermediates. The principal product from valine is propionylCoA,
the glucogenic precursor of succinyl-CoA.
 Isoleucine catabolism terminates with production of acetylCoA and
propionylCoA; thus isoleucine is both glucogenic and ketogenic. Leucine gives
rise to acetylCoA and acetoacetylCoA, and is thus classified as strictly ketogenic.
There are a number of genetic diseases associated with faulty catabolism of the
BCAAs. The most common defect is in the branched-chain α-keto acid
dehydrogenase.

Since there is only one dehydrogenase enzyme for all three amino acids, all
three α- keto acids accumulate and are excreted in the urine. The disease is known
as Maple syrup urine disease because of the characteristic odor of the urine in
afflicted individuals. Mental retardation in these cases is extensive. Unfortunately,
since these are essential amino acids, they cannot be heavily restricted in the diet;
ultimately, the life of afflicted individuals is short and development is abnormal
The main neurological problems are due to poor formation of myelin in the CNS.

Threonine Catabolism
 There are at least 3 pathways for threonine catabolism. One involves a
pathway initiated by threonine dehydrogenase yielding a-amino-b-ketobutyrate.
The α-amino-β-ketobutyrate is either converted to acetyl-CoA and glycine or
spontaneously degrades to aminoacetone which is converted to pyruvate. The
second pathway involves serine/threonine dehydratase yielding α-ketobutyrate
which is further catabolized to propionyl-CoA and finally the TCA cycle
intermediate, succinyl-CoA. The third pathway utilizes threonine aldolase. The
products of this reaction are both ketogenic (acetyl-CoA) and glucogenic
(pyruvate).

Methionine Catabolism

The principal fates of the essential amino acid methionine are incorporation
into polypeptide chains, and use in the production of α-ketobutyrate and cysteine
via SAM as described above. The transulfuration reactions that produce cysteine
from homocysteine and serine also produce α-ketobutyrate, the latter being
converted to succinyl-CoA. Regulation of the methionine metabolic pathway is
based on the availability of methionine and cysteine.
 If both amino acids are present in adequate quantities, SAM accumulates
and is a positive effector on cystathionine synthase, encouraging the production of
cysteine and α- ketobutyrate (both of which are glucogenic). However, if
methionine is scarce, SAM will form only in small quantities, thus limiting
cystathionine synthase activity. Under these conditions accumulated homocysteine
is remethylated to methionine, using N5-methylTHF and other compounds as
methyl donors.
 In 1968, a Harvard researcher observed that children with a genetic defect
that caused them to have sharply elevated homocysteine levels suffered severe
atherosclerotic occlusion and vascular disorders similar to what is seen in middle-
aged patients with arterial disease. This was the first indication that excess
homocysteine might be an independent risk factor for heart disease.

Cysteine Catabolism

There are several pathways for cysteine catabolism. The simplest, but least
important pathway is catalyzed by a liver desulfurase and produces hydrogen
sulfide, (H2S) and pyruvate. The more important catabolic pathway is via a
cytochrome-P450-coupled enzyme, cysteine dioxygenase that oxidizes the cysteine
sulfhydryl to sulfinate, producing the intermediate cysteinesulfinate.
Cysteinesulfinate can serve as a biosynthetic intermediate undergoing
decarboxylation and oxidation to produce taurine. Catabolism of cysteinesulfinate
proceeds through transamination to b-sulfinylpyruvate which is in undergoes
desulfuration yielding bisulfite, (HSO3-) and the glucogenic product, pyruvate. The
enzyme sulfite oxidase uses O2 and H2O to convert HSO3- to sulfate, (SO4-) and
H2O2. The resultant sulfate is used as a precursor for the formation of 3'-
phosphoadenosine-5'-phosphosulfate,PAPS.

PAPS is used for the transfer of sulfate to biological molecules such as the
sugars of the glycosphingolipids.Other than protein, the most important product of
cysteine metabolism is the bile salt precursor taurine, which is used to form the bile
acid conjugates taurocholate and taurochenodeoxycholate.The enzyme
cystathionase can also transfer the sulfur from one cysteine to another generating
thiocysteine and pyruvate. Transamination of cysteine yields -mercaptopyruvate
which then reacts with sulfite, (SO32-), to produce thiosulfate, (S2O32-) and
pyruvate. Both thiocysteine and thiosulfate can be used by the enzyme rhodanese
to incorporate sulfur into cyanide, (CN-), thereby detoxifying the cyanide to
thiocyanate.
 Phenylalanine and Tyrosine Catabolism

Phenylalanine normally has only two fates: incorporation into polypeptide

chains, and production of tyrosine via the tetrahydrobiopterin-requiring
phenylalanine hydroxylase. Thus, phenylalanine catabolism always follows the
pathway of tyrosine catabolism. The main pathway for tyrosine degradation
involves conversion to fumarate and acetoacetate, allowing phenylalanine and
tyrosine to be classified as both glucogenic and ketogenic. Tyrosine is equally
important for protein biosynthesis as well as an intermediate in the biosynthesis of
several physiologically important metabolites e.g. dopamine, norepinephrine and
epinephrine.

http://www.youtube.com/watch?v=hpaki7F4HR0

http://www.youtube.com/watch?v=CWfrVS4Bm1Y&feature=related

As in phenylketonuria (deficiency of phenylalanine hydroxylase), deficiency

of tyrosine transaminase leads to urinary excretion of tyrosine and the
intermediates between phenylalanine and tyrosine.
 Phenylketonuria (commonly known as PKU) is an inherited disorder that
increases the levels of a substance called phenylalanine in the blood. Phenylalanine
is a building block of proteins (an amino acid) that is obtained through the diet. It
is found in all proteins and in some artificial sweeteners. If PKU is not treated,
phenylalanine can build up to harmful levels in the body, causing intellectual
disability and other serious health problems.

The signs and symptoms of PKU vary from mild to severe. The most severe
form of this disorder is known as classic PKU. Infants with classic PKU appear
normal until they are a few months old. Without treatment, these children develop
permanent intellectual disability. Seizures, delayed development, behavioral
problems, and psychiatric disorders are also common. Untreated individuals may
have a musty or mouse-like odor as a side effect of excess phenylalanine in the
body. Children with classic PKU tend to have lighter skin and hair than unaffected
family members and are also likely to have skin disorders such as eczema.

Less severe forms of this condition, sometimes called variant PKU and non-
PKU hyperphenylalaninemia, have a smaller risk of brain damage. People with
very mild cases may not require treatment with a low-phenylalanine diet.
 Babies born to mothers with PKU and uncontrolled phenylalanine levels
(women who no longer follow a low-phenylalanine diet) have a significant risk of
intellectual disability because they are exposed to very high levels of phenylalanine
before birth. These infants may also have a low birth weight and grow more slowly
than other children. Other characteristic medical problems include heart defects or
other heart problems, an abnormally small head size (microcephaly), and
behavioral problems. Women with PKU and uncontrolled phenylalanine levels
also have an increased risk of pregnancy loss.

The adverse neurological symptoms are the same for the two diseases.
Genetic diseases (such as various tyrosinemias and alkaptonuria) are also
associated with other defective enzymes of the tyrosine catabolic pathway. The
first genetic disease ever recognized, alcaptonuria, is caused by defective
homogentisic acid oxidase. Homogentisic acid accumulation is relatively
innocuous, causing urine to darken on exposure to air, but no life-threatening
effects accompany the disease.
 The other genetic deficiencies lead to more severe symptoms, most of which
are associated with abnormal neural development, mental retardation, and
shortened life span.
 Albinism is a congenital disorder characterized by the complete or partial
absence of pigment in the skin, hair and eyes 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. While an organism with complete absence of
melanin is called an albino an organism with only a diminished amount of melanin
is described as albinoid.

Albinism is associated with a number of vision defects, such as photophobia,

nystagmus and astigmatism. Lack of skin pigmentation makes for more
susceptibility to sunburn and skin cancers. In rare cases such as Chédiak–Higashi
syndrome, albinism may be associated with deficiencies in the transportation of
melanin granules. This also affects essential granules present in immune cells
leading to increased susceptibility to infection.
 Lysine Catabolism

Lysine catabolism is unusual in the way that the α-amino group is

transferred to α-ketoglutarate and into the general nitrogen pool. The reaction is a
transamination in which the α-amino group is transferred to the α-keto carbon of α-
ketoglutarate forming the metabolite, saccharopine. Unlike the majority of
transamination reactions, this one does not employ pyridoxal phosphate as a
cofactor. Saccharopine is immediately hydrolyzed by the enzyme α-aminoadipic
semialdehyde synthase in such a way that the amino nitrogen remains with the α-
carbon of α-ketoglutarate, producing glutamate and α-aminoadipic semialdehyde.
Because this transamination reaction is not reversible, lysine is an essential amino
acid. The ultimate end-product of lysine catabolism is acetoacetyl-CoA Genetic
deficiencies in the enzyme α-aminoadipic semialdehyde synthase have been
observed in individuals who excrete large quantities of urinary lysine and some
saccharopine. The lysinemia and associated lysinuria are benign. Other serious
disorders associated with lysine metabolism are due to failure of the transport
system for lysine and the other dibasic amino acids across the intestinal wall.
Lysine is essential for protein synthesis; a deficiencies of its transport into the body
can cause seriously diminished levels of protein synthesis. Probably more
significant however, is the fact that arginine is transported on the same dibasic
amino acid carrier, and resulting arginine deficiencies limit the quantity of
ornithine available for the urea cycle. The result is severe hyperammonemia after a
meal rich in protein. The addition of citrulline to the diet prevents the
hyperammonemia. Lysine is also important as a precursor for the synthesis of
carnitine, required for the transport of fatty acids into the mitochondria for
oxidation. Free lysine does not serve as the precursor for this reaction, rather the
modified lysine found in certain proteins. Some proteins modify lysine to
trimethyllysine using SAM as the methyl donor to transfer methyl groups to the α -
amino of the lysine side chain. Hydrolysis of proteins containing trimethyllysine
provide the substrate for the subsequent conversion to carnitine.

Tryptophan Catabolism

A number of important side reactions occur during the catabolism of

tryptophan on the pathway to acetoacetate. The first enzyme of the catabolic
pathway is an iron porphyrin oxygenase that opens the indole ring. The latter
enzyme is highly inducible, its concentration rising almost 10-fold on a diet high in
tryptophan. Kynurenine is the first key branch point intermediate in the pathway.
Kynurenine undergoes deamniation in a standard transamination reaction yielding
kynurenic acid. Kynurenic acid and metabolites have been shown to act as
antiexcitotoxics and anticonvulsives. A second side branch reaction produces
anthranilic acid plus alanine. Another equivalent of alanine is produced further
along the main catabolic pathway, and it is the production of these alanine residues
that allows tryptophan to be classified among the glucogenic and ketogenic amino
acids. The second important branch point converts kynurenine into 2-amino-3-
carboxymuconic semialdehyde, which has two fates. The main flow of carbon
elements from this intermediate is to glutarate. An important side reaction in liver
is a transamination and several rearrangements to produce limited amounts of
nicotinic acid, which leads to production of a small amount of NAD+ and NADP+
Aside form its role as an amino acid in protein biosynthesis, tryptophan also serves
as a precursor for the synthesis of serotonin and melatonin.
 SPECIALIZED PRODUCTS OF AMINO ACIDS

Tyrosine-Derived Neurotransmitters

The majority of tyrosine that does not get incorporated into proteins is
catabolized for energy production. One other significant fate of tyrosine is
conversion to the catecholamines. The catecholamine neurotransmitters are
dopamine, norepinephrine, and epinephrine (see also Biochemistry of Nerve
Transmission).Norepinephrine is the principal neurotransmitter of sympathetic
postganglionic endings. Both norepinephrine and the methylated derivative,
epinephrine are stored in synaptic knobs of neurons that secrete it, however,
epinephrine is not a mediator at postganglionic sympathetic endings.Tyrosine is
transported into catecholamine-secreting neurons and adrenal medullary cells
where catechaolamine synthesis takes place. The first step in the process requires
tyrosine hydroxylase, which like phenylalanine hydroxylase requires
tetrahydrobiopterin as cofactor. The hydroxylation reaction generates DOPA (3,4-
dihydrophenylalanine). DOPA decarboxylase converts DOPA to dopamine,
dopamine b-hydroxylase converts dopamine to norepinephrine and
phenylethanolamine N-methyltransferase converts norepinephrine to epinephrine.
This latter reaction is one of several in the body that uses SAM as a methyl donor
generating S-adenosylhomocysteine. Within the substantia nigra and some other
regions of the brain, synthesis proceeds only to dopamine. Within the adrenal
medulla dopamine is converted to norepinephrine and epinephrine.
 Synthesis of the catecholamines from tyrosine

Once synthesized, dopamine, norepinephrine and epinephrine are packaged

in granulated vesicles. Within these vesicles, norepinephrine and epinephrine are
bound to ATP and a protein called chromogranin A. Metabolism of the
catecholemines occurs through the actions of catecholamine-O-methyltransferase,
(COMT) and monoamine oxidase, (MAO). Both of these enzymes are widley
distributed throughout the body. However, COMT is not found in nerve endings as
is MAO.

Tryptophan-Derived Neurotransmitters

Tryptopan serves as the precursor for the synthesis of serotonin (5-

hydroxytryptamine, 5-HT, see also Biochemistry of Nerve Transmission) and
melatonin (N-acetyl-5-methoxytryptamine).
 Serotonin is synthesized through 2-step process involving a
tetrahydrobiopterin-dependent hydroxylation reaction (catalyzed by tryptophan-5-
monooxygenase) and then a decarboxylation catalyzed by aromatic L-amino acid
decarboxylase. The hydroxylase is normally not saturated and as a result, an
increased uptake of tryptophan in the diet will lead to increased brain serotonin
content. Serotonin is present at highest concentrations in platelets and in the
gastrointestinal tract. Lesser amounts are found in the brain and the retina.
Serotonin containing neurons have their cell bodies in the midline raphe nuclei of
the brain stem and project to portions of the hypothalamus, the limbic system, the
neocortex and the spinal cord. After release from serotonergic neurons, most of the
released serotonin is recaptured by an active reuptake mechanism. The function of
the antidepressant, Prozac is to inhibit this reuptake process, thereby, resulting in
prolonged serotonin presence in the synaptic cleft. The function of serotonin is
exerted upon its interaction with specific receptors. Several serotonin receptors
have been cloned and are identified as 5HT1, 5HT2, 5HT3, 5HT4, 5HT5, 5HT6,
and 5HT7. Within the 5HT1 group there are subtypes 5HT1A, 5HT1B, 5HT1D,
5HT1E, and 5HT1F. There are three 5HT2 subtypes, 5HT2A, 5HT2B, and 5HT2C
as well as two 5HT5 subtypes, 5HT5a and 5HT5B. Most of these receptors are
coupled to G-proteins that affect the activities of either adenylate cyclase or
phospholipase C (PLC). The 5HT3 class of receptors are ion channels.Some
serotonin receptors are presynaptic and others postsynaptic. The 5HT2A receptors
mediate platelet aggregation and smooth muscle contraction. The 5HT2C receptors
are suspected in control of food intake as mice lacking this gene become obese
from increased food intake and are also subject to fatal seizures. The 5HT3
receptors are present in the gastrointestinal tract and are related to vomiting. Also
present in the gastrointestinal tract are 5HT4 receptors where they function in
secretion and peristalsis. The 5HT6 and 5HT7 receptors are distributed throughout
the limbic system of the brain and the 5HT6 receptors have high affinity for
antidepressant drugs. Melatonin is derived from serotonin within the pineal gland
and the retina, where the necessary N-acetyltransferase enzyme is found. The
pineal parenchymal cells secrete melatonin into the blood and cerebrospinal fluid.
Synthesis and secretion of melatonin increases during the dark period of the day
and is maintained at a low level during daylight hours. This diurnal variation in
melatonin synthesis is brought about by norepinephrine secreted by the
postganglionic sympathetic nerves that innervate the pineal gland. The effects of
norepinephrine are exerted through interaction with b-adrenergic receptors. This
leads to increased levels of cAMP, which in turn activate the N-acetyltransferase
required for melatonin synthesis. Melatonin functions by inhibiting the synthesis
and secretion of other neurotransmitters such as dopamine and GABA.

DISORDERS OF AMINO ACID METABOLISM

The figure below presents a diagram of pathways in which selected amino acids
are converted to citric acid cycle intermediates (and glucose) or to acetyl-CoA (and
ketones). Important genetic deficiencies are identified.

Phenylalanine Hydroxylase Deficiency (Phenylketonuria)

Infants with classic phenylketonuria (PKU) are normal at birth but if untreated
show slow development, severe mental retardation, autistic symptoms, and loss of
motor control. Children may have pale skin and white-blonde hair. The neurotoxic
effects relate to high levels of phenylalanine and not to the phenylketones from
which the name of the disease derives. Infants are routinely screened a few days
after birth for blood phenylalanine level. Treatment is a lifelong semisynthetic diet
restricted in phenylalanine (small quantities are necessary because it is an essential
amino acid).

Women with PKU who become pregnant must be especially careful about the
phenylalanine level in their blood so as not to adversely affect neurologic
development in the fetus. Infants whose phenylketonuric mothers have not
maintained adequate metabolic control during pregnancy have a high risk for
mental retardation (although less profound than in a child with untreated PKU),
microcephaly, and low birth weight.

Note Aspartame (N-aspartylphenylalanine methyl ester), a widely used artificial

sweetener, must be strictly avoided by phenylketonurics.

Bridge to Medical Genetics There are >100 known mutations in the gene for
phenylalanine hydroxylase, causing PKU. This is an example of allelic
heterogeneity.

Albinism
Albinism (1:15,000) is a group of conditions in which then normal conversion of
tyrosine to melanin is altered. The most severe form is a deficiency of tyrosinase,
causing an absence of pigment in the skin, hair, and eyes.

Homogentisate Oxidase Deficiency (Alcaptonuria)

Accumulation of homogentisic acid in the blood causes its excretion in urine, after
which it gradually darkens upon exposure to air. This sign of alcaptonuria is not
present in all patients with the enzyme deficiency. The dark pigment also
accumulates over years in the cartilage (ochronosis) and may be seen in the sclera
of the eye, in ear cartilage and patients develop arthritis in adulthood, usually
beginning in the third decade. Treatment is targeted to managing the symptoms

Branched-Chain Ketoacid Dehydrogenase Deficiency (Maple Syrup Urine

Disease)

Branched-chain ketoacid dehydrogenase, an enzyme similar to α-ketoglutarate

dehydrogenase (thiamine, lipoic acid, CoA, FAD, NAD+), metabolizes
branchedchain ketoacids produced from their cognate amino acids, valine, leucine,
and isoleucine. In the classic form of the disease, infants are normal for the first
few days of life, after which they become progressively lethargic, lose weight, and
have alternating episodes of hypertonia and hypotonia, and the urine develops a
characteristic odor of maple syrup. Ketosis, coma, and death ensue if not treated.
Treatment requires restricting dietary valine, leucine, and isoleucine.

Propionyl-CoA Carboxylase and Methylmalonyl-CoA Mutase Deficiencies

Valine, methionine, isoleucine, and threonine are all metabolized through the
propionic acid pathway (also used for odd-carbon fatty acids). Deficiency of either
enzyme results in neonatal ketoacidosis from failure to metabolize ketoacids
produced from these 4 amino acids. The deficiencies may be distinguished based
on whether methylmalonic aciduria is present (methylmalonyl CoA mutase
deficiency) or by the presence of methyl citrate and hydroxypropionate (propionyl
CoA carboxylase deficiency). A diet low in protein or a semisynthetic diet with
low amounts of valine, methionine, isoleucine, and threonine is used to treat both
deficiencies

Note

• Propionyl CoA carboxylase deficiency involves an accumulation of propionic

acid, methyl citrate, and hydroxypropionic acid.

• Methylmalonyl CoA mutase deficiency involves an accumulation of

methylmalonic acid.

Homocystinemia/Homocystinuria

Accumulation of homocystine in blood is associated with cardiovascular disease;

deep vein thrombosis, thromboembolism, and stroke; dislocation of the lens
(ectopic lens); and mental retardation. Homocystine is a disulfide dimer of
homocysteine. Homocystinemia caused by an enzyme deficiency is a rare, but
severe, condition in which atherosclerosis in childhood is a prominent finding.
These children often have myocardial infarctions before 20 years of age. All
patients excrete high levels of homocystine in the urine. Treatment includes a diet
low in methionine. The major enzyme deficiency producing homocystinemia is
that of cystathionine synthase:

A 5-year-old girl was brought to her pediatrician because she had difficulty with
her vision and seemed to be slow in her mental and physical development since
birth. The physician noted that the girl had abnormally long, “spidery” fingers
and a downward dislocation of the right lens of her eye. Further examination
revealed a deep vein thrombosis. A laboratory examination of her blood indicated
increased methionine. She also had increased urinary excretion of homocystine,
indicated by a cyanidenitroprusside test. The parents were advised to restrict
methionine to low levels and supplement folate, vitamin B12, and vitamin B6 in the
girl’s diet. Homocystinuria caused by a genetic defect in the enzyme cystathionine
synthase is rare and can present similarly to Marfan syndrome. The latter is a
defect in the fibrillin gene, resulting in tall stature, long fingers and toes, lens
dislocation, and a tendency toward aortic wall ruptures. In cystathionine synthase
deficiency subluxation of the lens is downward and inward. In Marfan syndrome
subluxation of the lens is upward and outward.

Cystathionine synthase deficiency results in the accumulation of homocysteine and

methionine and their spillage into blood and urine. Two molecules of
homocysteine can oxidize to the disulfide-crosslinked homocystine. Many patients
with homocystinuria who have partial activity of cystathionine synthase respond
well to pyridoxine administration. If left untreated, patients will usually succumb
to myocardial infarction, stroke, or pulmonary embolism.

Homocystinemia from Vitamin Deficiencies

Vitamin deficiencies may produce a more mild form of homocystinemia. Mild

homocystinemia is associated with increased risk for atherosclerosis, deep vein
thrombosis, and stroke.

The vitamin deficiencies causing homocystinemia include:

• Folate deficiency: recommended dietary intake of folate has been increased (also
protects against neural tube defects in the fetus), and additional folate is now
added to flour (bread, pasta, and other products made from flour)

• Vitamin B12

• Vitamin B6

S-ADENOSYLMETHIONINE, FOLATE, AND COBALAMIN

One-Carbon Units in Biochemical Reactions

One-carbon units in different oxidation states are required in the pathways

producing purines, thymidine, and many other compounds. When a biochemical
reaction requires a methyl group (methylation), S-adenosylmethionine (SAM) is
generally the methyl donor. If a 1-carbon unit in another oxidation state is required
(methylene, methenyl, formyl), tetrahydrofolate (THF) typically serves as its
donor.

S-Adenosylmethionine Important pathways requiring SAM include synthesis of

epinephrine and of the 7-methylguanine cap on eukaryotic mRNA. After donating
the methyl group, SAM is converted to homocysteine and remethylated in a
reaction catalyzed by N-methyl THF–homocysteine methyltransferase requiring
both vitamin B12 and N-methyl-THF. The methionine produced is once again used
to make SAM

Note THB (BH4) is necessary for tyrosine hydroxylase, phenylalanine

hydroxylase, and tryptophan hydroxylase (serotonin synthesis) and is regenerated
by dihydropteridine reductase.

Tetrahydrofolate

THF is formed from the vitamin folate through 2 reductions involving NADPH
and catalyzed by dihydrofolate reductase. It picks up a 1-carbon unit from a variety
of donors and enters the active 1-carbon pool. Important pathways requiring forms
of THF from this pool include the synthesis of all purines and thymidine, which in
turn are used for DNA and RNA synthesis during cell growth and division.
Megaloblastic anemia results from insufficient active THF to support cell division
in the bone marrow. Methotrexate inhibits DHF reductase, making it a useful
antineoplastic drug.

Folate deficiencies may be seen during pregnancy and in alcoholism. Additional

folate may be stored as the highly reduced N5-methyl-THF. This form is referred
to as the storage pool as there is only one known enzyme that uses it, and in turn
moves it back into the active pool. This enzyme is N-methyl THF-homocysteine
methyltransferase, which also requires vitamin B12 and is involved in regenerating
SAM as a methyl donor for reactions.
Clinical Correlate Parkinson’s disease is caused by loss of dopaminergic neurons
in the substantia nigra. It is treated with levodopa (L-dopa).

Cobalamin

The vitamin cobalamin (vitamin B12) is reduced and activated in the body to 2
forms: adenosylcobalamin (used by methylmalonyl-CoA mutase) and
methylcobalamin (formed from N5-methyl-THF in the N-methyl THF-
homocysteine methyltransferase reaction).

These are the only 2 enzymes which use B12 (other than the enzymes that reduce
and add an adenosyl group to it).

Cobalamin deficiency can result in the following:

• Secondary deficiency of active THF, by preventing its release from the storage
pool through the N-methyl THF-homocysteine methyltransferase reaction (thus
also results in megaloblastic anemia)

• Progressive peripheral neuropathy

• Pernicious anemia (most likely reason), a failure to absorb vitamin B12 in the
absence of intrinsic factor from parietal cells

• B12 absorption also decreases with aging and in individuals with chronic
pancreatitis.

• B12 absorption can also decrease (less commonly) with a long-term, completely
vegetarian diet (plants don’t contain B12) or infection with Diphyllobothrium
latum, a parasite found in raw fish (excess B12 is stored in the body, so
deficiencies develop slowly) Treating a cobalamin deficiency with folate corrects
the megaloblastic anemia but does not halt the neuropathy.

Table 2. Folate versus Vitamin B12 Deficiency

Folate Deficiency Vitamin B12 (Cobalamin) Deficiency

Megaloblastic anemia ● Macrocytic

● Macrocytic ● MCV >100 femtoliters (fL)

● MCV >100 femtoliters (fL) ● PMN nucleus more than 5 lobes

Homocysteinemia with risk for
● PMN nucleus more than 5 lobes
cardiovascular disease
Homocysteinemia with risk for
cardiovascular disease Deficiency develops in 3–4 months
Risk factors for deficiency:
Deficiency develops in 3–4 months
Risk factors for deficiency: ● Pregnancy (neural tube defects in
fetus may result)
● Pregnancy (neural tube defects in
fetus may result) ● Alcoholism

● Alcoholism ● Severe malnutrition

● Severe malnutrition ● Gastric or terminal ileum resection

● Gastric or terminal ileum resection Megaloblastic anemia

● Macrocytic

● MCV >100 femtoliters (fL)

● PMN nucleus more than 5 lobes

Homocysteinemia with risk for
cardiovascular disease
Methylmalonic aciduria Progressive
peripheral neuropathy Deficiency
develops in years

Risk factors for deficiency:

● Pernicious anemia
 ● Gastric resection

● Chronic pancreatitis

● Severe malnutrition

● Vegan

● Infection with D. latum

● Aging

● Bacterial overgrowth of the

terminal ileum

● H. pylori infection

SPECIALIZED PRODUCTS DERIVED FROM AMINO ACIDS

Table 3. Products of Amino Acids

Amino Acid Products

Tyrosine Thyroid hormones T3 and T4 Melanin

Catecholamines

Tryptophan Serotonin NAD, NADP

Arginine Nitric oxide (NO)

Glutamate γ-Aminobutyric acid (GABA)

Histidine Histamine

Creatine Biosynthesis
 Creatine is synthesized in the liver by methylation of guanidoacetate using
SAM as the methyl donor. Guanidoacetate itself is formed in the kidney from the
amino acids arginine and glycine.

Synthesis of creatine and creatinine

 Creatine is used as a storage form of high energy phosphate. The phosphate
of ATP is transferred to creatine, generating creatine phosphate, through the action
of creatine phosphokinase. The reaction is reversible such that when energy
demand is high (e.g. during muscle exertion) creatine phosphate donates its
phosphate to ADP to yield ATP.Both creatine and creatine phosphate are found in
muscle, brain and blood. Creatinine is formed in muscle from creatine phosphate
by a nonenzymatic dehydration and loss of phosphate. The amount of creatinine
produced is related to muscle mass and remains remarkably constant from day to
day. Creatinine is excreted by the kidneys and the level of excretion (creatinine
clearance rate) is a measure of renal function.

Glutathione Functions

Glutathione (abbreviated GSH) is a tripeptide composed of glutamate,

cysteine and glycine that has numerous important functions within cells. It serves
as a reductant, is conjugated to drugs to make them more water soluble, is involved
in amino acid transport across cell membranes (the γ-glutamyl cycle), is a part of
the peptidoleukotrienes, serves as a cofactor for some enzymatic reactions and as
an aid in the rearrangement of protein disulfide bonds.
 Synthesis of glutathione (GSH) Structure of GSSG

The role of GSH as a reductant is extremely important particularly in the

highly oxidizing environment of the erythrocyte. The sulfhydryl of GSH can be
used to reduce peroxides formed during oxygen transport. The resulting oxidized
form of GSH consists of two molecules disulfide bonded together (abbreviated
GSSG).

The enzyme glutathione reductase utilizes NADPH as a cofactor to reduce

GSSG back to two moles of GSH. Hence, the pentose phosphate pathway is an
extremely important pathway of erythrocytes for the continuing production of the
NADPH needed by glutathione reductase. In fact as much as 10% of glucose
consumption, by erythrocytes, may be mediated by the pentose phosphate pathway.
Several mechanisms exist for the transport of amino acids across cell membranes.
Many are symport or antiport mechanisms that couple amino acid transport to
sodium transport. The γ-glutamyl cycle is an example of a group transfer
mechanism of amino acid transport. Although this mechanism requires more
energy input, it is rapid and has a high capacity. The cycle functions primarily in
the kidney, particularly renal epithelial cells. The enzyme γ-glutamyl
transpeptidase is located in the cell membrane and shuttles GSH to the cell surface
to interact with an amino acid. Reaction with an amino acid liberates
cysteinylglycine and generates a γ-glutamyl-amino acid which is transported into
the cell and hydrolyzed to release the amino acid. Glutamate is released as 5-
oxoproline and the cysteinylglycine is cleaved to its component amino acids.
Regeneration of GSH requires an ATP-dependent conversion of 5-oxoproline to
glutamate and then the 2 additional moles of ATP that are required during the
normal generation of GSH.

Polyamnine Biosynthesis

One of the earliest signals that cells have entered their replication cycle is
the appearance of elevated levels of mRNA for ornithine decarboxylase (ODC),
and then increased levels of the enzyme, which is the first enzyme in the pathway
to synthesis of the polyamines. Because of the latter, and because the polyamines
are highly cationic and tend to bind nucleic acids with high affinity, it is believed
that the polyamines are important participants in DNA synthesis, or in the
regulation of that process.
 The key features of the pathway are that it involves putrescine, an ornithine
catabolite, and S-adenosylmethionine (SAM) as a donor of 2 propylamine residues.
The first propylamine conjugation yields spermidine and addition of another to
spermidine yields spermine.The function of ODC is to produce the 4-carbon
saturated diamine, putrescine. At the same time, SAM decarboxylase cleaves the
SAM carboxyl residue, producing decarboxylated SAM (S-
adenosymethylthiopropylamine), which retains the methyl group usually involved
in SAM methyltransferase activity. SAM decarboxylase activity is regulated by
product inhibition and allosterically stimulated by putrescine. Spermidine synthase
catalyzes the condensation reaction, producing spermidine and 5'-
methylthioadenosine. A second propylamine residue is added to spermidine
producing spermine.The signal for regulating ODC activity is unknown, but since
the product of its activity, putrescine, regulates SAM decarboxylase activity, it
appears that polyamine production is principally regulated by ODC
concentration.The butylamino group of spermidine is used in a posttranslational
modification reaction important to the process of translation. A specific lysine
residue in the translational initiation factor eIF-4D is modified. Following the
modification the residue is hydroxylated yielding a residue in the protein termed
hypusine.

Nitric Oxide Synthesis and Function

Vasodilators, such as acetylcholine, do not exert their effects upon the

vascular smooth muscle cell in the absence of the overlying endothelium. When
acetylcholine binds its receptor on the surface of endothelial cells, a signal cascade,
coupled to the activation phospholipase C (PLC), is initiated. The PLCg-mediated
release of inositol trisphosphate, IP3 (from membrane associated
phosphatidylinositol-4,5-bisphosphate, PIP2), leads to the release of intracellular
stores of Ca2+. In turn, the elevation in Ca2+ leads to the liberation of endothelium-
derived relaxing factor (EDRF) which then diffuses into the adjacent smooth
muscle. Within smooth muscle cells, EDRF reacts with the heme moiety of a
soluble guanylyl cyclase, resulting in activation of the latter and a consequent
elevation of intracellular levels of cGMP. The net effect is the activation of cGMP-
responsive enzymes which lead to smooth muscle cell relaxation. The coronary
artery vasodilator, nitroglycerin, acts to increase intracellular release of EDRF and
thus of cGMP. Quite unexpectedly, EDRF was found to be the free radical
diatomic gas, nitric oxide, NO. NO is formed by the action of NO synthase, (NOS)
on the amino acid arginine.

Nitric oxide is involved in a number of other important cellular processes in

addition to its impact on vascular smooth muscle cells. Events initiated by NO that
are important for blood coagulation include inhibition of platelet aggregation and
adhesion and inhibition of neutrophil adhesion to platelets and to the vascular
endothelium. NO is also generated by cells of the immune system and as such is
involved in non-specific host defense mechanisms and macrophage-mediated
killing. NO also inhibits the proliferation of tumor cells and microorganisms.
Additional cellular responses to NO include induction of apoptosis (programmed
cell death), DNA breakage and mutation. NOS is a very complex enzyme,
employing five redox cofactors: NADPH, FAD, FMN, heme and
tetrahydrobiopterin. NO can also be formed from nitrite, derived from vasodilators
such as glycerin trinitrate during their metabolism. The half-life of NO is
extremely short, lasting only 2-4 seconds. This is because it is a highly reactive
free radical and interacts with oxygen and superoxide. NO is inhibited by
hemoglobin and other heme proteins which bind it tightly.Chemical inhibitors of
NOS are available and can markedly decrease production of NO. The effect is a
dramatic increase in blood pressure due to vasoconstriction. Another important
cardiovascular effect of NO is exerted through the production of cGMP, which acts
to inhibit platelet aggregation.