Urea Cycle

• Role of Urea cycle: to prevent the accumulation of toxic NH 4+. It

incorporates nitrogen not used for biosynthetic purposes into urea,
which serves as the waste nitrogen product in mammals.

• Urea is the major disposal form of amino groups derived from

amino acids, and accounts for about 90% of the nitrogen-containing
components of urine.

• Also urea cycle is responsible for de novo synthesis of arginine.

• The complete Urea Cycle is significantly only in liver.

• However some enzymes of the pathway are in other cells and

tissues where they generate arginine & ornithine.

• E.g., Argininosuccinate Synthase, which catalyzes synthesis of the

precursor to arginine, is in most tissues.
 cytosol

Reactions of the cycle mitochondrial matrix

carbamoyl phosphate
Pi
ornithine citrulline

ornithine citrulline
urea aspartate
arginine argininosuccinate
fumarate

• 5-step Urea Cycle (Small Krebs c., Krebs-Hensleit c., Ornitihin c.)
• function: synthesis of non-toxic urea.
• The first two reactions leading to the synthesis of urea occur in the mitochondria,
whereas the remaining cycle enzymes are located in the cytosol.
• One nitrogen of the urea molecule is supplied by free NH3, and the other nitrogen
by aspartate.
• So glutamate is the immediate precursor of both ammonia (through oxidative
deamination by glutamate dehydrogenase) and aspartate nitrogen (through
transamination of oxaloacetate by AST).
• The carbon and oxygen of urea are derived from CO2.
• Overall urea cycle reaction:

• Urea is produced by the liver, and then is transported in the blood to the kidneys
for excretion in the urine.
Step 1: formation of Carbamoyl phosphate from
ammonia, bicarbonate and ATP
• Enzyme Carbamoyl phosphate synthetase I.
• In liver cell mitochondrias.
• Ammonia released from the oxidative deamination of glutamate, by
mitochondrial glutamate dehydrogenase, is incorporated in
carbamoyl phosphate by using ATP and bicarbonate.
• Utilizes 2 ATP .
• Requires Mg+
• Carbamoyl phosphate synthetase I requires N-acetylglutamate as a
positive allosteric activator. The reaction is irreversible.
 HCO3−
ATP
• Carbamoyl Phosphate ADP
Synthase (Type I) catalyzes O
a 3-step reaction, with 2−
HO C OPO3
carbonyl phosphate and carbonyl phosphate
NH3
carbamate intermediates. Pi
• Carbamoyl phosphate O
synthetase (type)II H2N C O−
participates in the ATP carbamate
biosynthesis of pyrimidines, ADP
It does not require N- O
acetylglutamate, and occurs H2N C OPO3 2−
in the cytosol. carbamoyl phosphate
 Step 2: Formation of citrulline from ornithine
and carbamoyl phosphate
• Enzyme Ornithine
transcarbamoylase
• In liver mitochondria
• Citrulline is formed from transfer
of the carbamoyl group to the γ-
amino group of ornithine.
• The release of the high-energy
phosphate of carbamoyl
phosphate as inorganic phosphate
drives the reaction in the forward
direction.
• Citrulline passes from
mitochondrial membrane to
cytosol
• Ornithine is regenerated with each
turn of the urea cycle.
 Step 3: Formation of arginosuccinate from
citrulline and aspartate
• This is the second nitrogen-acquiring reaction.
• Condensation of citrulline with aspartate to form arginosuccinate.
• The α-amino group of aspartate provides the second nitrogen that is
ultimately incorporated into urea.
• Enzyme Argininosuccinate synthetase.
• Requires ATP and Mg+
 Step 4: Formation of arginine and
fumarate from arginosuccinate
• Enzyme Argininosuccinase
• Liver and kidney of mammals
• Cleaves arginosuccinate to form arginine and
fumarate.
• The arginine formed by this reaction serves as
the immediate precursor of urea.
• Fumarate produced in the urea cycle is
hydrated to malate, providing a link with
several metabolic pathways. For example, the
malate can be transported into the
mitochondria via the malate shuttle and
reenter the tricarboxylic acid cycle.
Alternatively, cytosolic malate can be oxidized
to oxaloacetate, which can be converted to
aspartate or glucose. ((thus aspartate could
be regenerated)
 Step 5: Hydrolysis of arginine to form ornithine
and urea
• Enzyme Arginase
• The arginine is hydrolyzed to produce the urea and to reform the
ornithine.
• In liver cells cytosol.
• The ornithine reenters the mitochondrial matrix.
• Urea excreted (N rich, excelent solubility, harmless )
• Thus, whereas other tissues, such as the kidney, can synthesize
arginine by these reactions, only the liver can cleave arginine and,
thereby, synthesize urea.
 Fate of urea
• Urea diffuses from the liver, and is transported in the blood
to the kidneys, where it is filtered and excreted in the urine.
• A portion of the urea diffuses from the blood into the
intestine, and is cleaved to CO2 and NH3 by bacterial
urease.
• This ammonia is partly lost in the feces, and is partly
reabsorbed into the blood.
• In patients with kidney failure, plasma urea levels are
elevated, promoting a greater transfer of urea from blood
into the gut.
• The intestinal action of urease on this urea becomes a
clinically important source of ammonia, contributing to the
hyperammonemia often seen in these patients.
 Regulation of urea cycle
• The Activity of the Urea Cycle Is Regulated at Two Levels :the level of
urea cycle enzyme synthesis and by allosteric regulation of the enzyme
carbamoyl phosphate synthetase I.
• The flux of nitrogen through the urea cycle in an individual animal varies
with diet. When the dietary intake is primarily protein, the carbon
skeletons of amino acids are used for fuel, producing much urea from the
excess amino groups.
• During prolonged starvation, when breakdown of muscle protein begins to
supply much of the organism’s metabolic energy, urea production also
increases substantially.
• These changes in demand for urea cycle activity are met over the long
term by regulation of the rates of synthesis of the enzymes in the liver.
• All five enzymes are synthesized at higher rates in starving animals and in
animals on very-high-protein diets than in well-fed animals eating
primarily carbohydrates and fats.
• Animals on protein-free diets produce lower levels of urea cycle enzymes.
Regulation of urea cycle cont..:
•The carbamoyl phosphate
synthetase I, is allosterically
activated by N-acetylglutamate,
which is synthesized from
acetyl- CoA and glutamate by N-
acetylglutamate synthase.
•The steady-state levels of N-
acetylglutamate are determined
by the concentrations of
glutamate and acetyl-CoA (the
substrates for N-acetylglutamate
synthase) and arginine(an
activator of N-acetylglutamate
synthase, and thus an activator
of the urea cycle).
urea cycle
Aspartate -Arginosuccinate Shunt Links Urea Cycle
and Citric Acid Cycle
 Aspartate -Arginosuccinate Shunt
Links Urea Cycle and Citric Acid Cycle
• Each cycle can operate independently and communication between them
depends on the transport of key intermediates between the
mitochondrion and cytosol.
• Several enzymes of the citric acid cycle, including fumarase (fumarate
hydratase) and malate dehydrogenase, are also present as isozymes in the
cytosol.
• The fumarate generated in cytosolic arginine synthesis can therefore be
converted to malate in the cytosol, and these intermediates can be further
metabolized in the cytosol or transported into mitochondria for use in the
citric acid cycle.
• Aspartate formed in mitochondria by transamination between
oxaloacetate and glutamate can be transported to the cytosol, where it
serves as nitrogen donor in the urea cycle reaction catalyzed by
argininosuccinate synthetase.
 Hyperammonemia
• The capacity of the hepatic urea cycle exceeds the normal rates of
ammonia generation, and the levels of serum ammonia are
normally low (5-50 µmol/L).
• When liver function is decreased, due either to genetic defects of
the urea cycle, or liver disease, blood levels can rise above 1,000
µmol/L.
• Ammonia has a direct neurotoxic effect on the CNS. For example,
elevated concentrations of ammonia in the blood cause the
symptoms of ammonia intoxication, which include tremors, slurring
of speech, sleepy, vomiting, cerebral edema, and blurring of vision.
At high concentrations, ammonia can cause coma and death.
• The two major types of hyperammonemia are:Acquired
hyperammonemia Hereditary hyperammonemia
 Acquired Hyperammonemia
• Liver disease is a common cause of hyperammonemia in
adults.
• Liver diseases include viral hepatitis, ischemia, or
hepatotoxins.
• In patients with liver disease, hepatic function is impaired
and conversion ammonia to urea, therefore, severely
impaired, leading to elevated levels of circulating ammonia.
• Cirrhosis of the liver caused by alcoholism, hepatitis or
biliary obstruction may result in formation of collateral
circulation around the liver.
• As a result, portal blood is shunted directly into the
systemic circulation and does not have access to the liver.
 Hereditary hyperammonemia
Urea Cycle Disorders
• Deficiency or absence of any one of urea cycle enzymes resulting
in one urea cycle disorder .
• Hyperammoniemia is the characteristic and predominant cause of
the symptoms.
• Complete deficiency of any enzyme of them leads to severe
hyperammoniemia in the neonatal period and may result in coma
and death .
• Partial enzyme deficiency leads to clinical picture correlated with
the residual enzyme activity and the age of onset of the disease .
• In some cases the hyperammoniemia is episodic and this episodes
can result from high protein diet or infections .
• Prevalence of disorders: 1/30,000 live births but may be more
since some die undiagnosed.
 Symptoms

Severe Illness: First week

Usually normal first 24h
Symptoms of hyperammonemia within 1-3 days
Include: Feeding intolerance
Vomiting
Lethargy
Irritability
Respiratory Distress (hyperventilation)
Seizures
Coma
 Hyperammoniemia Toxicity
• Hyperammoniemia cause encephalopthy ( brain damage ) mainly by these
mechanisms :
• 1- Shift of glutamate dehydrogenase reaction toward the direction of glutamate
formation .
• The resulting depletion of α-ketoglutarate, an essential Krebs Cycle intermediate,
could impair energy metabolism in the brain.
• 2- Increased glutamine also cause brain damage .( Fig )
• 3- This would deplete glutamate - a neurotransmitter & precursor for synthesis of
the neurotransmitter GABA.

Glutamate
dehydrogenase
NADH NAD+
α-Ketoglutarate + Glutamate
NH3 + ATP
NH4 +
Glutamine
synthetase

ADP + Pi
Glutamine
Diagrammatic representation of the pathophysiology responsible for hyperammonemic
encephalopathy. The primary event is activation by ammonia of glutamine synthetase within the
astrocyte, leading to the accumulation of glutamine within the astrocyte. This has a dual effect: (1)
glutamine serves as an intracellular osmolyte, causing entry of water and astrocyte swelling and
cerebral edema, and (2) the swollen astrocyte or the high glutamine concentration causes astrocyte
dysfunction.
Urea Cycle Disorders
 Treatment of Urea Cycle Disorders
1. limiting protein intake to the amount
barely adequate to supply amino acids for
growth, while adding to the diet the α-
keto acid analogs of essential amino
acids.
2. Feeding the patients with Benzoate or
phenylacectate: These compound react
with glycine and glutamine respectively
forming non-toxic compounds that are
excreted in urine. Thus the body runs
low in glycine and glutamine and starts
synthasizing these AA using the ammonia
available in system. Thus clearing the
system of excess ammonia.
3. In the patients with N-acetylglutamate
synthase deficiency, Carbamoyl glutamate
(NAG Analog) can act as activator of
carbamoyl phosphate synthase.
4. Liver transplantation has also been used, since
liver is the organ that carries out Urea Cycle.