The Urea Cycle (Ornithine Cycle)

Overview

The urea cycle is the primary pathway for disposing of nitrogen from amino acid catabolism. Urea (H₂N-CO-NH₂) accounts for ~90% of the nitrogen-containing components of urine. It was first described in 1932 by Hans Krebs and Kurt Henseleit - hence the original name, the Krebs-Henseleit cycle.

One nitrogen comes from free NH₃ (ammonia)
The other nitrogen comes from aspartate
The carbon and oxygen come from CO₂ (as HCO₃⁻)
The cycle occurs in hepatocytes of the liver
Steps 1-2 occur in the mitochondrial matrix; steps 3-5 occur in the cytosol

Urea is released into the blood (measured as BUN - blood urea nitrogen) and excreted by the kidneys.

The 5 Reactions

Step 1 - Carbamoyl Phosphate Synthesis (Mitochondria)

Enzyme: Carbamoyl phosphate synthetase I (CPS I)

NH₃ + HCO₃⁻ + 2 ATP → Carbamoyl phosphate + 2 ADP + Pi

Requires cleavage of 2 ATP
Requires N-acetylglutamate (NAG) as an obligatory allosteric activator
This is the rate-limiting, committed step
Note: CPS II (cytosolic) is a different enzyme that participates in pyrimidine synthesis; it uses glutamine as nitrogen source and does not require NAG

Step 2 - Citrulline Formation (Mitochondria)

Enzyme: Ornithine transcarbamylase (OTC)

Carbamoyl phosphate + Ornithine → Citrulline + Pi

The carbamoyl group is transferred to ornithine
Citrulline exits the mitochondria via an antiporter (exchanges with ornithine)
OTC deficiency is the most common urea cycle disorder (X-linked)

Step 3 - Argininosuccinate Formation (Cytosol)

Enzyme: Argininosuccinate synthetase (ASS)

Citrulline + Aspartate + ATP → Argininosuccinate + AMP + PPi

This step introduces the second nitrogen (from aspartate)
Requires ATP hydrolysis to AMP + PPi (equivalent to 2 ATP)

Step 4 - Cleavage of Argininosuccinate (Cytosol)

Enzyme: Argininosuccinate lyase (ASL)

Argininosuccinate → Arginine + Fumarate

Arginine retains both nitrogens
Fumarate is released - it enters the TCA cycle (connects urea cycle to TCA), eventually being converted back to oxaloacetate → aspartate via transamination (the "aspartate-argininosuccinate shunt")

Step 5 - Urea Release (Cytosol)

Enzyme: Arginase I

Arginine + H₂O → Urea + Ornithine

Urea is released and excreted
Ornithine is regenerated and transported back into the mitochondria to restart the cycle (analogous to oxaloacetate in the TCA cycle)

Energy Cost

Step	ATP equivalent used
CPS I (Step 1)	2 ATP → 2 ADP + Pi
ASS (Step 3)	ATP → AMP + PPi (= 2 ATP equivalent)
Total	4 ATP per urea molecule

Regulation

The key regulatory enzyme is CPS I, controlled by:

N-acetylglutamate (NAG) - obligatory allosteric activator
NAG is synthesized from acetyl-CoA + glutamate by NAG synthase
Arginine activates NAG synthase - so when arginine (and therefore protein load) is high, the cycle speeds up
High protein intake and prolonged fasting both increase urea cycle enzyme expression

Link to the TCA Cycle

The fumarate released in Step 4 connects to the TCA cycle:

Fumarate → Malate (fumarase) → Oxaloacetate (malate dehydrogenase) → Aspartate (transamination with glutamate)

This regenerates aspartate for Step 3, creating a cyclic interplay between the urea cycle and TCA cycle.

Nitrogen Sources - How NH₃ and Aspartate Are Generated

Glutamate dehydrogenase (GDH): Glutamate → α-ketoglutarate + NH₃ (feeds Step 1)
Aspartate aminotransferase (AST): Oxaloacetate + Glutamate → Aspartate + α-ketoglutarate (feeds Step 3)
Glutamate is therefore the immediate precursor of both nitrogen atoms in urea

Hyperammonemia

Normal blood ammonia: 5-35 μmol/L (some sources give 30-60 μM). When liver function is compromised, levels can exceed 1,000 μmol/L.

Symptoms: Tremors, slurred speech, somnolence, vomiting, cerebral edema, coma, and death.

Causes:

Acquired - liver disease (viral hepatitis, alcohol-induced cirrhosis, portal-systemic shunting bypasses hepatic urea synthesis)
Inherited (urea cycle disorders):

Disorder	Deficient Enzyme	Key Features
CPS I deficiency	Carbamoyl phosphate synthetase I	↑ NH₃, no orotic acid
OTC deficiency	Ornithine transcarbamylase	Most common; X-linked; ↑ orotic acid (carbamoyl-P diverted to pyrimidine synthesis)
Citrullinemia	Argininosuccinate synthetase	↑ citrulline
Argininosuccinic aciduria	Argininosuccinate lyase	↑ argininosuccinate
Arginase deficiency	Arginase I	↑ arginine; spastic diplegia
NAGS deficiency	N-acetylglutamate synthase	↑ NH₃; treated with N-carbamylglutamate

Key distinguishing point: OTC deficiency produces elevated orotic acid in urine (excess carbamoyl phosphate in the cytosol is shunted into the pyrimidine synthesis pathway), while CPS I deficiency does not.

Treatment of Hyperammonemia

Nitrogen scavengers: Sodium benzoate (conjugates glycine → hippurate, excreted) and sodium phenylbutyrate (→ phenylacetate → conjugates glutamine → phenylacetylglutamine, excreted) - both allow nitrogen excretion without urea
Dietary protein restriction and essential amino acid supplementation
Arginine supplementation (except in arginase deficiency) - arginine becomes essential when the cycle is blocked
Liver transplant for severe defects
N-carbamylglutamate (a NAG analog) for NAGS deficiency - activates residual CPS I

Sources: Biochemistry, 8th ed. Lippincott Illustrated Reviews, p. 712-722; Basic Medical Biochemistry - A Clinical Approach, 6e, p. 1321-1326

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