Biochemistry Ch 38 707-721
The Urea Cycle
Fate of Amino Acid Nitrogen
1. Transamination Reactions – removes nitrogen from amino acids, whereby the N is
transferred as an amino group to an α-ketoglutarate to form glutamate
-original amino acid converted to α-ketoacid
-all AA EXCEPT lysing + arginine can undergo transaminations
-done by transaminases
-pyridoxal phosphate is REQUIRED COFACTOR, derived from vitamin B6, activation:
-Pyridoxine (B6)  Pyridoxaldehyde  Pyridoxal Phosphate (PLP)
-Vitamin B6 deficiency – dermatitis, microcytic, hypochromic anemia. Xanthurenic acid appears
in urine because of inability to metabolize amino acids, decreased ability to synthesize heme
from glycine causes hypochromic anemia
2. Removal of Amino Acid Nitrogen as Ammonia – cells release nitrogen from AA as ammonia
or ammonium. Physiologic pH favors ammonium ion by factor of 100/1
-NH3 also present because it can cross membranes
-Glutamate can be oxidatively deaminated by glutamate dehydrogenase to produce
NH4 and α-ketoglutarate (Glutamate  NH4 + α-ketoglutarate) using NAD or NADP in
mitochondria. Readily reversible and ammonium can be interconverted
-Many other AA can lose nitrogen as NH4+
-Histidine  Urocanate
-Serine/Threonine deaminations require pyridoxal phosphate catalyzed by serine
dehydratase
-Glutamine and Arginine have R groups can be released as NH4
-Asparagine deamidated by asparaginase yielding aspartate and NH4
-Glutaminase acts on glutamine, important in kidney where NH4 excreted
-in muscle/brain, purine nucleotide cycle allows NH4 to be released from AA
-glutamate collects N and transfers it to oxaloacetate to form aspartate which supplies
nitrogen to purine nucleotide cycle, which releases fumarate and NH4
3. Role of Glutamate in Metabolism of Amino Acid Nitrogen – glutamate involved in synthesis
and degradation of amino acids
-provides N for AA synthesis, and obtains its own nitrogen by transamination reactions
or from NH4 by glutamate dehydrogenase
-When AA are degraded, glutamate collects N from other amino acids by transamination
reactions, some of which is released by ammonia by glutamate dehydrogenase and
march larger amounts of ammonia are produced from other sources
-NH4 is the one of the main forms in which nitrogen enters urea cycle
-second form of N for urea synthesis is aspartate, from which glutamate is source of N
4. Role of Alanine and Glutamine in Transporting AA Nitrogen to Liver – urea cycle enzymes
are primarily in the liver, mechanism is in place to transport AA N to liver
-Alanine/glutamine are major transporters of nitrogen in blood
-Alanine is exported primarily by muscle; pyruvate  alanine by transamination through
glutamate, which then travels to liver
-in liver, alanine is transaminated to pyruvate, and N is used for urea synthesis while
pyruvate is used for gluconeogenesis while glucose exported to muscle for energy
-cycling of N between liver and muscle called glucose/alanine cycle
-glutamine synthesized from glutamate by fixation of ammonia, ATP, and glutamine
synthetase in cells around portal vein
-under conditions of rapid AA breakdown, ammonia increases, and glutamate that has
been formed from transamination accepts another nitrogen to form glutamine, which
travels to liver, kidney, or intestines where glutaminase removes amide to form
glutamate + ammonia
Urea Cycle – major nitrogen product is urea found in urine; it serves as a disposal of ammonia
which is toxic to the CNS.
-urea cycle was proposed, called Krebs-Henseleit cycle
Reactions of the Urea Cycle – nitrogen enters the cycle as NH4 and aspartate to form carbamoyl
phosphate, which reacts with ornithine to form citrulline
-ornithine initiates and is regenerated by the cycle
-aspartate reacts with citrulline to donate its nitrogen to urea after forming arginine
-cleavage of arginine by arginase releases urea and regenerates ornithine
CO2 + H2O  HCO3 + NH4 Carbamoyl Phosphate + Ornithine  Citrulline  Aspartate 
Argininosuccinate  Arginine  Ornithine + Urea
1. Synthesis of Carbamoyl Phosphate – NH4, HCO3 + ATP react to form carbamoyl phosphate
with the help of carbamoyl phosphate synthetase (CPSI)
2. Production of Arginine by Urea Cycle – in the mitochondria, carbamoyl phosphate reacts
with ornithine to form citrulline and is catalyzed by ornithine transcarbamoylase (OTC)
-product is transported across mit. membrane in exchange for cytoplasmic ornithine
-citrulline reacts with aspartate in cytoplasm  argininosuccinate (argininosuccinate
synthetase) driven by ATPAMP + P2 and aspartate comes from deamination of oxaloacetate
-Argininosuccinate cleaved by argininosuccinate lyase  fumarate and arginine
-fumarate converted to malate used for gluconeogenesis or for regeneration of
oxaloacetate by TCA cycle
-oxaloacetate that is formed is transaminated to aspartate which carries N to urea cycle
***Clinical correlate – ornithine transcarbamoylase deficiency causes carbamoyl phosphate to
accumulate and flood pyrimidine biosynthesis instead of enter urea cycle, causing an excess
amount of orotic acid (orotate) to be excreted in urine with no ill effects
3. Cleavage of Arginine to Produce Urea – Arginine, containing N from NH4 and aspartate, is
cleaved by arginase to produce urea and regenerate ornithine
-urea is produced from the guanidinium group on side chain of arginine
-ornithine is transported back into the mitochondria in exchange for citrulline
Origin of Ornithine – ornithine is an amino acid not incorporated into protein synthesis because
it has no codon. Apart from being regenerated by urea cycle, ornithine can be synthesized de
novo catalyzed by ornithine aminotransferase in the intestine, converting glutamate
semialdehyde to ornithine in the first step
Regulation of Urea Cycle – urea cycle is regulated by substrate availability; the higher the rate of
ammonia production, the higher is the rate of urea formation (regulation by substrate
availability is common among disposal pathways, and is a common feed-forward regulation)
-allosteric activation of CSPI by N-acetylglutamate (NAG) positively regulates urea cycle. NAG is
only formed to induce CPSI and has no known functions otherwise
-NAG is formed from acetyl-coA and glutamate and is stimulated by arginine
-Glutamate + Acetyl-CoA  N-Acetylglutamate (induced by arginine)
-as arginine increases in liver, two reactions are stimulated
1. Synthesis of N-acetylglutamate, which increases rate of carbamoyl phosphate
production
2. Production of more ornithine via arginase reaction so that cycle can be rapid
-Urea cycle also regulated by induction of urea-cycle enzymes in response to conditions that
require increased protein metabolism such as high-protein diet or prolonged fasting, where in
both conditions amino acid nitrogen is converted to urea
Function of Urea Cycle During Fasting – during fasting, liver maintains blood glucose, and amino
acids from muscle protein are a major carbon source for production of glucose by
gluconeogenesis
-as AA carbons converted to glucose, nitrogens are converted to urea, this urinary
excretion of urea is high during fasting
-as fasting proceeds, brain starts to use ketone bodies, and not so much glucose is
needed, so less muscle protein is cleaved for gluconeogenesis which causes decreased
production of urea
-major amino acid substrate for gluconeogenesis is alanine, which is synthesized in peripheral
tissues to act as a nitrogen carrier
-glucagon release during fasting stimulates alanine transport into liver by activating
transcription of transport systems for alanine
-2 molecules of alanine  glucose, and the nitrogen of 2 alanines  1 urea
-NH4+ is a toxin that results from degradation of urea or proteins by intestinal bacteria and is
not metabolized by the infected liver
-elevation of NH4 in brain fluid causes depletion of TCA intermediates and ATP in CNS
-α-ketoglutarate combines with NH3 to form glutamate by glutamate dehydrogenase
and glutamate + NH3  glutamine
-GABA (gamma aminobutyric acid), an inhibitory neurotransmitter is shunted into
systemic circulation from gut lumen in patients with hepatic failure
-In addition to producing urea, reactions of TCA biosynthesize arginine, making arginine not an
essential AA in the diet, but urea cannot be cleaved by humans because Urease is only produced
by bacteria
-1/4th of all urea released by liver each day is recycled by bacteria
Disorders of Urea Cycle – accumulation of ammonia is dangerous in circulation, as it is toxic to
CNS, so levels in the body must be controlled.
-free ammonia is fixed into α-ketoglutarate or glutamate (through glutamate dehydrogenase to
form glutamate and glutamine synthetase to form glutamamine, respectively).
-glutamine can be used by liver; as nitrogens removed from carriers, CPSI fixes NH3 into
carbamoyl phosphate to initiate urea cycle
-when urea cycle enzyme is defective, cycle is interrupted and intermediates
accumulate
-if there is a block, glutamine levels rise in the blood, and because a-ketoglutarte levels
are too low to fix free ammonia, elevated ammonia is found in the blood
-any urea-cycle enzyme defect causes glutamine accumulation and high ammonia in the blood
-Ammonia toxicity will lead to brain swelling due to osmotic imbalance caused by high levels of
ammonia and glutamine in astrocytes
-as ammonia levels rise in astrocytes, more glutamine is produced to exacerbate
imbalance
-ammonia levels inhibit glutaminase to lead to glutamine elevation
-high levels of glutamine alter permeability of mitochondrial membrane to open the
mitochondrial permeability transition pore, which leads to cell death
-absence of glutamate is due to high ammonia, which causes brain dysfunction since glutamate
is a neurotransmitter, can cause lethargy and CNS activity reduction
-most common urea-cycle defect is OTC deficiency, X-linked disorder
-major clinical problem with urea-cycle defects in the effect of excessive blood ammonia on
CNS, which can lead to irreversible neuronal damage and mental retardation
-low-protein diets re essential to reduce potential for excessive amino acid degradation
-drugs are used to form conjugates with amino acids, most frequent are benzoic acid and
phenylbutyrate
-phenylacetate has a bad odor. Phenylactete forms a conjugate with glutamine which is
excreted and removed two nitrogens per molecule
-benzoic acid, after activation reacts with glycine to form hippuric acid which is excreted
-as glycine is synthesized from serine, body now uses nitrogens to synthesize serine, so
more glycine can be produced
-gene therapy experiments can be used on people with OTC deficiencies