Nitrogen Metabolism

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Nitrogen Metabolism
Digestion of protein and the release of free
amino acids Transamination of amino acids The
urea cycle and the excretion of nitrogenous waste
products Breakdown of amino acid carbon skeletons
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Learning Objectives Identify the nonessential,
conditionally essential, and essential amino
acids in human metabolism. Describe conditionally
essential for arginine, tyrosine, and
cysteine. Describe the three major circumstances
in which amino acids undergo oxidative
degradation. Describe the digestion of
proteins. Describe the secretion of HCl by the
parietal cells. Define achlorhydria. Describe the
function of the proteases and peptidases of the
small intestine. Describe the activation of the
intestinal proteases and peptidases.
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Learning Objectives Write the structural
equation for the transamination of an amino
acid. Identify the function of glutamate,
glutamine, and alanine with respect to the amino
group in nitrogen metabolism. Describe and/or
diagram the glucose-alanine cycle. Describe the
reactions catalyzed by glutamine synthase,
glutaminase, glutamate dehydrogenase, alanine
aminotransferase. Describe the urea
cycle. Describe nitrogen balance. Draw the
structure of pyridoxal phosphate draw the
structure of pyridoxal phosphate as a Schiff-base
with lysine. Describe the regulation of the urea
cycle.
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Learning Objectives Describe the degradation of
the branched chain amino acids. Describe what is
meant by a ketogenic or glucogenic amino
acid. Describe the urea cycle.
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Amino Acids
Mammals synthesize the nonessential amino acids
from metabolic precursors but must obtain the
essential amino acids from their diet. Amino
acids are protein monomeric units, and precursors
for many biologically important
nitrogen-containing compounds heme,
physiologically active amines, glutathione, and
nucleotides.
Excess dietary amino acids are neither stored for
future use nor excreted. They are converted to
common metabolic intermediates such as pyruvate,
oxaloacetate, and a-ketoglutarate. Consequently,
amino acids are precursors of glucose, fatty
acids, and ketone bodies and are therefore
metabolic fuels.
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Nonessential and essential amino acids for humans
Nonessential
Conditionally Essential
Essential
Required to some degree in young, growing
animals, and/or sometimes during illness.
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Children and pregnant women have a high rate of
protein synthesis to support growth, and require
some arginine in their diet although it can be
synthesized in the body.
Histidine is essential in the diet of adults in
very small quantities because adults efficiently
recycle histidine.
Tyrosine is synthesized from phenylalanine, and
it required in the diet if phenylalanine intake
is inadequate.
Cysteine is synthesized by using sulfur from
methionine, and may be required if methionine
intake is low.
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In animals, amino acids undergo oxidative
degradation in three different metabolic
circumstances
(1) During the normal degradation of cellular
proteins (protein turnover), if the amino acids
are not needed for new protein synthesis. (2)
When a diet is rich in protein and the ingested
amino acids exceed the bodys need for protein
synthesis, the surplus is catabolized. Amino
acids cannot be stored. (3) When carbohydrates
are either unavailable or not properly utilized
(starvation or diabetes), cellular proteins are
degraded and the amino acids used as fuel.
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Overview of amino acid catabolism in mammals
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General structure of an amino acid
carboxylic acid group
amino group
The R group or side chain attached to the
a-carbon is different in each amino acid
This structure is common to all but one of the
standard a-amino acids. The cyclic amino acid,
proline, is the exception.
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Amino acids are linked by peptide bonds to form
proteins
peptide bond (shaded)
Enzymes called proteases catalyze the hydrolytic
cleavage of peptide bonds
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Gastric glands
Parietal cells (secrete HCl)
Portion of the human digestive tract
Chief cells (secrete pepsinogen)
Stomach
Low pH
Exocrine cells of pancreas
Pancreas
Rough endoplasmic reticulum (ER)
Pancreatic duct
Zymogen granules
Collecting duct
pH 7
Villi of small intestine
Villus
Small intestine
Intestinal mucosa
(absorbs amino acids)
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Gastric glands in the stomach lining
The parietal cells and chief cells secrete their
products in response to the hormone gastrin,
which is secreted by the gastric mucosa upon
entry of dietary protein into the stomach.
Parietal cells secrete acidic (HCl) gastric juice
(pH 1.0 to 2.5) which is both an antiseptic and a
denaturing agent, unfolding globular proteins and
rendering their internal peptide bonds more
accessible to enzymatic hydrolysis.
Chief cells secrete pepsinogen, an inactive
precursor or zymogen. This is converted to the
active enzyme pepsin by the action of pepsin
itself. In the stomach, pepsin hydrolyzes
peptide bonds on the amino-terminal side of the
aromatic amino acids phenylalanine, tyrosine, and
tryptophan, cleaving long polypeptide chains into
mixtures of smaller peptides.
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Secretion of HCl by the parietal cells of the
stomach
The H for HCl is produced by carbonic anhydrase
H2CO3
CO2 H2O
H2CO3
H CO3-
carbonic anhydrase
Protons are secreted by the parietal cells
against a concentration gradient (10-7 M versus
10-1 M). This is accomplished by a P-type
transporter, the (H- K)-ATPase.
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Secretion of HCl by the parietal cells of the
stomach
Electroneutral cotransport of K with Cl-
Electroneutral H-K antiport
Extracellular
pH 1-2
Cl-
K
H
Plasma membrane
K
Intracellular
ATP
ADP Pi
pH 7
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Achlorhydria
In approximately 4 of the total adult population
and 40 of those older than 60 years, HCL
secretion by the stomach is absent. This
condition is known as achlorhydria. In these
individuals, pepsin is not active (pepsin has a
pH optimum of 2) and digestion of protein does
not begin until food passes into the intestine.
There are a number of proteases in the intestine,
and usually no major clinical symptoms result.
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pH change between stomach and small intestine
As the acidic stomach contents pass into the
small intestine, the low pH triggers secretion of
the hormone secretin into the blood. Secretin
stimulates the pancreas to secrete bicarbonate
into the small intestine to neutralize the
gastric HCl. All secretions from the pancreas
into the small intestine pass through the
pancreatic duct.
Exocrine cells of pancreas the term exocrine
denotes secretions outside the body as through
ducts that lead to the gastrointestinal tract.
(Endocrine denotes internal secretions of
biologically active substances either into the
blood or into the interstitial fluid between
cells).
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Proteases and peptidases of the small intestine
Arrival of amino acids in the upper part of the
intestine (duodenum) causes release into the
blood of the hormone cholecystokinin, which
stimulates the release of several pancreatic
enzymes into the small intestine
trypsinogen chymotrypsinogen procarboxypeptidase
A and B
These are the zymogen forms of
trypsin chymotrypsin carboxypeptidase
A and B
zymogen an inactive precursor of an enzyme
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Activation of trypsinogen
Enteropeptidase is a proteolytic enzyme secreted
by intestinal cells.
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Activation of chymotrypsinogen
Trypsin then converts additional trypsinogen into
active trypsin, and also activates
chymotrypsinogen, the procarboxypeptidases, and
proelastase.
Peptides A, B, and C in a-chymotrypsin are linked
by disulfide bonds.
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Trypsin and chymotrypsin have an active site
serine residue, and are called serine
proteases. Trypsin (cleaves at carboxyl side of
arginine and lysine residues) and chymotrypsin
(cleaves at carboxyl side of phenylalanine,
tyrosine, and tryptophan residues) further
hydrolyze the peptides produced by pepsin in the
stomach. Other intestinal peptidases carboxypept
idases A and B (both Zn-containing enzymes )
remove successive amino acids from the carboxyl
end of peptides aminopeptidase hydrolyzes
successive residues from the amino-terminal end
of peptides
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Trypsin cleaves peptide bonds on the carbonyl (or
C-terminal) side of arginines and lysines
H3N-Ala-Gly-Asp-Leu-Arg-Ile-Gly-Phe-Tyr-Lys-Ala-Al
a-COOH
trypsin
H3N-Ala-Ala-COOH
H3N-Ala-Gly-Asp-Leu-Arg-COOH
H3N-Ile-Gly-Phe-Tyr-Lys-COOH
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H3N-Ala-Gly-Asp-Leu-Arg-Ile-Gly-Phe-Tyr-Ile-Ala-Al
a-COOH
H2O
H3N-Ala-Gly-Asp-Leu-Arg-COOH
H3N-Ile-Gly-Phe-Tyr-Ile-Ala-Ala-COOH
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Cleavage of amino acids by carboxypeptidase
H3N-Ala-Trp-Gly-Leu-Ile-Ala-Arg-COOH

H3N-Ala-Trp-Gly-Leu-Ile-Ala-COOH
Arg

H3N-Ala-Trp-Gly-Leu-Ile-COOH
Ala

H3N-Ala-Trp-Gly-Leu-COOH
Ile
Continued cleavage of C-terminal amino acids
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Absorption of free amino acids
The sequential action of these proteases and
peptidases yields a mixture of free amino acids
that can be transported into the epithelial cells
lining the small intestine. The free amino acids
enter the blood capillaries in the villi and
travel to the liver and other tissues in the body.
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Overview catabolism of amino acids in mammals
Every amino acid contains an amino group. The
a-amino group is separated from the carbon
skeleton and shunted into the pathway of amino
group metabolism. The remaining carbon skeleton
is degraded or utilized in biosynthetic reactions.
Intracellular Protein
Dietary Protein
Amino Acids
Carbon Skeletons
NH4
Biosynthesis of amino acids, nucleotides, and
biological amines
a-Keto acids
Carbamoyl phosphate
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Metabolic fates of amino groups
Nitrogen, N2, is abundant in the atmosphere but
only a few microorganisms can convert N2 to
biologically useful forms such as NH3. Therefore
amino groups are carefully handled in biological
systems. Amino acids derived from dietary protein
are the source of most amino groups. Most amino
acids are oxidized in the liver. Some of the
derived ammonia is recycled in other biosynthetic
pathways. In most terrestrial vertebrates, the
excess ammonia is converted to urea for
excretion. Excess ammonia generated in
non-hepatic tissues is transported to the liver
(in the form of amino groups) for either
recycling or excretion.
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Cellular protein
Amino acids from ingested protein
Liver
a-Keto acids
Amino acids
Glutamine from muscle and other tissues
Glutamate
a-Ketoglutarate
Alanine from muscle
Urea
(for excretion)
Alanine
Pyruvate
Glutamine
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Transamination of L-amino acids
The first step in the degradation of amino acids
is the removal of the amino group catalyzed by
enzymes called aminotransferases or transaminases.
The a-amino group is transferred to the a-carbon
of a-ketoglutarate. There is no net deamination
(loss of amino group). The amino acid is
converted to an a-keto acid.
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Function of L-glutamate
The effect of the transamination reactions is to
collect the amino groups from many different
amino acids in the form of L-glutamate.
Glutamate then functions as an amino group donor
for biosynthetic pathways or excretion pathways
that lead to the elimination of nitrogenous waste
products.
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Aminotransferases and pyridoxal phosphate
Cells contain different aminotransferases many
differ in their specificity of the L-amino acid.
All aminotransferases have the same prosthetic
group pyridoxal phosphate (PLP).
Pyridoxal phosphate is one of the coenzyme forms
of vitamin B6, pyridoxine.
HO
HO
CH2
Pyridoxine
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Schiff-base formation
Pyridoxal phosphate is bound to transaminases
(and other enzymes) through a Schiff-base linkage
with the e-amino group of a lysine at the active
site.
PLP functions as an intermediate carrier of amino
groups in the active site of aminotransferases.
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Ammonia is a toxic substance in animals
Ammonia is produced by the cellular degradation
of amino acids and nucleic acids in all tissues
as part of normal metabolic processes. However,
only the liver and kidneys can convert ammonia to
urea for excretion. Other tissues must be able
to dispose of free ammonia and transport it to
the liver in some nontoxic form.
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Free ammonia is combined with glutamate to yield
glutamine by the enzyme glutamine
synthase. Glutamine is a nontoxic transport
form of ammonia it is normally present in blood
in much higher concentrations than any other
amino acid.
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Glutamine, in excess of that required for
biosynthesis is transported in the blood to the
liver and kidneys for processing.
The amide nitrogen is released as ammonia only in
the mitochondria of liver and kidney by the
enzyme glutaminase.
L-glutamine
L-glutamate
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The Cori Cycle
The circulatory systems of large animals,
including humans, cannot sustain aerobic
metabolism in skeletal muscle during long bursts
of muscular activity. Stores of muscle glycogen
are rapidly depleted by glycolysis to produce
ATP. The pyruvate is fermented to lactate which
builds up to high levels in the blood. It is
slowly converted back to glucose by
gluconeogenesis in the liver in the subsequent
rest or recovery period during which oxygen is
consumed at a gradually diminishing rate until
the breathing rate returns to normal. This
oxygen consumed during the recovery period
represents a repayment of the oxygen debt.
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The Cori Cycle
glycogen
Muscle ATP produced by glycolysis and fermented
to lactate under anaerobic conditions
glucose
lactate
ATP
The cycle of reactions that includes glucose
conversion to lactate in muscle and lactate
conversion to glucose in liver is called the Cori
cycle.
Blood lactate
Blood glucose
Liver ATP used in synthesis of glucose
(gluconeogenesis) during recovery from strenuous
muscle activity.
lactate
glucose
ATP
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In skeletal muscle and other tissues that degrade
amino acids for fuel, amino groups are collected
in the form of glutamate by transamination. Glutam
ate can acquire additional ammonia to form
glutamine, or it can transfer its a-amino group
to pyruvate by the enzyme alanine
aminotransferase.
pyruvate
glutamate
alanine aminotransferase
alanine
a-ketoglutarate
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The glucose-alanine cycle
Vigorously contracting skeletal muscle operate
anaerobically, producing pyruvate and lactate via
glycolysis as well as ammonia from protein
breakdown. These products must find their way to
the liver, where pyruvate and lactate are
converted into glucose, which is returned to the
muscles, and ammonia is converted into urea for
excretion.
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Glutamate releases ammonia in the liver
On a high protein diet, there is an excess of
amino groups derived from the amino acids. These
temporarily accumulate in glutamate.
In the liver, glutamate is transported into the
mitochondria where it undergoes oxidative
deamination catalyzed by the enzyme L-glutamate
dehydrogenase.
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Oxidative deamination of glutamate by glutamate
dehydrogenase
In mammals, this enzyme is present in the
mitochondria and can utilize either NAD or NADP
as electron acceptor. This is an important
intersection of carbon and nitrogen metabolism.
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Glutamate dehydrogenase is an allosteric enzyme
with six identical subunits. Its activity is
influenced by a complicated array of allosteric
modulators. The best-studied are the positive
modulator ADP and the negative modulator GTP.
The metabolic rational for this regulatory
pattern has not been elucidated. Mutations that
alter the GTP binding site (causing permanent
activation of glutamate dehydrogenase) lead to a
human genetic disorder called hyperinsulinism-hype
rammonemia syndrome. This is characterized by
increased levels of ammonia in the blood, and
hypoglycemia (low blood glucose).
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Excess ammonia is deposited in the mitochondria
of liver for excretion.
Alanine
(from muscle)
Glutamine
(from extrahepatic tissues)
Amino acids
Glutamate
Glutamine
glutaminase
Glutamate
NH4
glutamate dehydrogenase
a-ketoglutarate
Liver Mitochondrial Matrix
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The Urea Cycle
The ammonia deposited in the mitochondria of
hepatocytes is converted to urea in the urea
cycle. Urea production occurs almost exclusively
in the liver and is the fate of most of the
ammonia channeled there. The urea passes into the
blood stream and thus to the kidneys and is
excreted into the urine.
The urea cycle begins in the mitochondria, but
most of the reactions occur in the cytosol the
cycle thus spans two cellular compartments.
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(1) ornithine transcarbamoylase
(2) argininosuccinate synthetase
(3) argininosuccinate lyase
(4) arginase
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The NH4 generated in the liver mitochondria is
immediately used, together with CO2 (as HCO3-)
produced by mitochondrial respiration, to form
carbamoyl phosphate in the matrix.
This reaction is catalyzed by carbamoyl phosphate
synthetase I. (The cytosolic form (II) of this
enzyme functions in pyrimidine biosynthesis).
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PPi
The Urea Cycle
ATP
Citrullyl-AMP intermediate
Aspartate
(2)
AMP
Citrulline
2 ATP
2 ADP
Urea Cycle
Pi
Argininosuccinate
(1)
Carbamoyl phosphate
NH4
(3)
Ornithine
HCO3-
Fumarate
Arginine
Ornithine
H2O
(4)
(1) ornithine transcarbamoylase
Urea
(2) argininosuccinate synthetase
O
(3) argininosuccinate lyase
H2N
NH2
C
(4) arginase
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The citrulline transported out of the
mitochondria is not diluted into the general pool
of metabolites in the cytosol, but is passed
directly to the active site argininosuccinate
synthase. This channeling between enzymes
continues and only urea is released into the
general pool of metabolites in the cytosol.
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Regulation of urea cycle
The flux of nitrogen through the urea cycle
depends upon diet.
Primarily protein diet carbon skeletons of
amino acids are used for fuel, and a lot of urea
is generated from the excess ammonia.
Prolonged starvation muscle protein is broken
down to supply energy and urea production
increases.
Long term regulation involves rates of synthesis
of the enzymes of the urea cycle and carbamoyl
phosphate synthetase I in the liver.
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Mammals synthesize N-acetyl glutamate which is an
allosteric activator of carbamoyl phosphate
synthetase I. (This enzyme catalyzes the first
step in the de novo synthesis of arginine from
glutamate in plants and microorganisms. Mammals
lack the other enzymes in this pathway. Thus, the
use of this compound in mammals is enigmatic).
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Nitrogen Balance
When the amount of nitrogen ingested in the diet
equals the amount excreted in the urine, the
individual is said to be in nitrogen balance.
A positive nitrogen balance occurs when there is
a net gain in the amount of nitrogen in the body.
This occurs in growth, healing, and pregnancy.
In negative nitrogen balance, there is a net loss
of body nitrogen. Since there is no mechanism
for storing nitrogen, this is detrimental to
health. A negative nitrogen balance can occur in
malnutrition, after surgery, and in the elderly,
especially if they are confined to bed.
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Genetic Defects
Individuals with defects in any of the urea cycle
enzymes cannot tolerate protein-rich diets. An
excess of ammonia is produced in the liver that
cannot be converted to urea. This ammonia is
exported into the blood stream where it can be
toxic.
Hyperammonemia
Defect in carbamoyl phosphate synthetase
Type I
Hyperammonemia
Defect in ornithine transcarbamoylase
Type II
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Careful administration of benzoate in the diet
can help lower the blood ammonia levels. The
regeneration of glycine uses up ammonia.
benzoate
NH4
(as glutamate)
benzoyl-CoA
3-phosphoglycerate
glycine
Hippurate is a nontoxic compound that can be
excreted in the urine.
hippurate (benzoylglycine)
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More effective because it utilizes glutamine, one
of the major carriers of ammonia.
phenylacetate
NH4
phenylacetyl-CoA
glutamine
glutamate
glutamine synthetase
phenylacetylglutamine
(nontoxic excreted in urine)
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Pathways of Amino Acid Degradation
The pathways of amino acid catabolism normally
account for only 10 to 15 of the human bodys
energy production. Hence, these pathways are not
nearly as active as glycolysis and fatty acid
oxidation.
Flux through the individual pathways varies
greatly depending upon biosynthetic requirements
and the availability of a specific amino acid.
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The 20 catabolic pathways converge to form only
five products which enter the TCA cycle.
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Tetrahydrofolate
The oxidized form, folate, is a vitamin for
mammals and is converted to tetrahydrofolate by
the enzyme dihydrofolate reductase. This cofactor
is involved in one-carbon transfers in amino acid
and nucleotide metabolism.
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S-Adenosylmethionine
S-Adenosylmethionine is the preferred cofactor
for biological methyl group transfers. It is
synthesized from ATP and methionine (an essential
amino acid) by the action of methionine adenosyl
transferase the first step in methionine
degradation).
This cofactor is also important in amino acid and
nucleotide metabolism.
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Branched-chain amino acids are not degraded in
the liver
Although much of the catabolism of amino acids
takes place in the liver, the three amino acids
with branched side chains (valine, leucine, and
isoleucine) are oxidized as fuels primarily in
muscle, kidney, adipose, and brain tissue. These
extahepatic tissues contain an aminotransferase,
absent in liver, that acts on all three
branched-chain amino acids to produce the
corresponding a-ketoacids. The branched-chain
a-ketoacid dehydrogenase complex then catalyzes
the oxidative decarboxylation of all three
a-ketoacids to produce the acyl-CoA derivatives.
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Maple syrup urine disease
There is a relatively rare genetic disease in
which the three branched-chain a-ketoacids (as
well as the precursor amino acids, especially
leucine) accumulate in the blood and spill over
into the urine. This condition, called maple
syrup urine disease because of the characteristic
odor imparted to the urine by the a-ketoacids,
results from a defective branch-chain a-ketoacid
dehydrogenase complex. Untreated, the disease
results in abnormal development of the brain,
mental retardation, and death in early infancy.
Treatment entails rigid control of diet, limiting
the intake of valine, leucine, and isoleucine to
the minimum required to permit normal growth.
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Glucogenic and ketogenic amino acids
These are the ketogenic amino acids because they
can yield ketone bodies in the liver. This is
particularly evident in untreated diabetes in
which the liver produces large amounts of ketone
bodies from both fatty acids and the ketogenic
amino acids.
Tryptophan Phenylalanine Tyrosine Isoleucine
Leucine Lysine
degradation
ketone bodies (in the liver)
Acetoacetyl-CoA and/or Acetyl-CoA
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ketogenic blue
glucogenic red
67
The division between glucogenic and ketogenic
amino acids is not sharp.
Tryptophan Phenylalanine Tyrosine Isoleucine
Lysine
These amino acids are both glucogenic and
ketogenic
Leucine is an exclusively ketogenic amino acid
that is very common in proteins. Its degradation
makes a substantial contribution to ketosis under
starvation conditions.
(Lysine is considered as both glucogenic and
ketogenic, but its glucogenic intermediate is
unknown).
The remaining amino acids are glucogenic.
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Genetic defects in amino acid metabolism
Many genetic defects of amino acid metabolism
have been identified in humans. In most of these
diseases, specific intermediates accumulate (are
present in the urine) and cause defective neural
development and mental retardation.
Phenylketonuria (PKU) is an inherited defect in
phenylalanine metabolism in which phenylalanine
hydroxylase is the defective enzyme. This enzyme
produces tyrosine from phenylalanine.
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Phenylalanine hydroxylase
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Alternate pathways for catabolism of
phenylalanine in phenylketonuria
Phenylalanine and phenylpyruvate accumulate in
tissues, blood, and are found in the urine. Much
of the phenylpyruvate is converted to
phenylacetate or phenyllactate which is excreted.