Question

Biochemistry.

What is the key substrate that nitrogen goes onto to enter the Urea Cycle?

Answer 1

Alanine and glutamine are the major transporters of nitrogen in the blood. Alanine is produced in a single biochemical step by the transamination of pyruvate. Glutamine is produced from glutamate by the addition of an amide to the glutamate γ carboxyl group by an ATP-dependent reaction catalyzed by glutamine synthetase.

NH₄⁺ and aspartate, the forms in which nitrogen enters the urea cycle, are produced from amino acids in the liver by a series of transamination and deamination reactions. Glutamate dehydrogenase is a key enzyme in the process because it generates the free NH₄⁺ previously transferred to α-ketoglutarate from many amino acids by transaminases. As dietary protein increases (a protein-rich diet) the concentration of the enzymes of the urea cycle increase, suggesting a regulated response to meet the increased need for nitrogen disposal.

Reactions of the Urea Cycle

Two nitrogen atoms enter the urea cycle as NH₄⁺ and aspartate. The first steps of the cycle take place in liver mitochondria, where NH₄⁺ combines with HCO₃^- to form carbamoyl phosphate. Carbamoyl phosphate reacts with ornithine, a compound both required as input to, and regenerated by the cycle, to produce citrulline, which, exits the mitochondria to the cytosol, where the remaining reactions of the cycle occur. The amino acid arginine is synthesized as a product of the urea cycle. Fumarate, another product, links the urea cycle with the TCA cycle. The two entering nitrogen atoms exit the cycle as urea, which the liver releases into the blood for disposal, in urine, by the kidneys.

1. Synthesis of carbamoyl phosphate by Carbamoyl Phosphate Synthetase I
   • in mitochondria of the liver
   • one of only three reactions in humans that can “fix” NH₄⁺, i.e., covalently link it to carbon
   • NH₄⁺, CO₂ (as bicarbonate) and 2 ATP react to form carbamoyl phosphate.
   • 2 ATP molecules provide the energy to create the phosphoanhydride and N-C bonds of carbamoyl phosphate; inorganic phosphate and 2 ADP produced.
   • stimulated by N-acetyl-glutamate (a required allosteric activator), which is synthesized from acetyl CoA and glutamate; the synthesis of N-acetyl-glutamate is stimulated by arginine, the immediate precursor of urea in the urea cycle. Increased levels of amino acids, signaled by increased arginine levels, therefore, stimulate urea production by the urea cycle.
   • NOTE: Carbamoyl phosphate synthetase I is present in liver mitochondria and uses NH₄⁺ as a source of nitrogen; carbamoyl phosphate synthetase II is present in the cytosol of many cells, uses glutamine as a source of nitrogen, and produces carbamoyl phosphate for pyrimidine biosynthesis.
2. Synthesis of citrulline from carbamoyl phosphate and ornithine by Ornithine Transcarbamoylase
   • X-linked gene
   • in mitochondria; ornithine transported into mitochondria
   • carbamoyl phosphate is the carbamoyl donor which has a high transfer potential because of its phosphoanhydride bond
   • inorganic phosphate released
   • citrulline produced, which is transported from the mitochondria to the cytosol where the remaining reactions of the urea cycle occur
3. Synthesis of argininosuccinate by condensation of citrulline and aspartate by Argininosuccinate Synthetase
   • driven by the cleavage of ATP; AMP and inorganic pyrophosphate produced; inorganic pyrophosphate cleaved by cellular pyrophosphatases to inorganic phosphate
4. Argininosuccinate cleaved by Argininosuccinase to produce fumarate and arginine
   • NOTE: The carbon skeleton of aspartate is conserved as fumarate, with transfer of the aspartate amino group to arginine. Recall that fumarate is a TCA cycle intermediate, and can be hydrated to form malate. In the fed state malate may be converted by malic enzyme to pyruvate, which serves as a source for the synthesis of fatty acids. It may also be oxidized to oxaloacetate. Oxaloacetate can have several fates. It can be transaminated to aspartate (aspartate transaminase), combine with acetyl CoA to enter the TCA cycle or, in the starved state, be converted to phosphoenolpyruvate for gluconeogenesis.
5. Urea production and the regeneration of ornithine from arginine by Arginase
   • urea passes into the blood and is eliminated by the kidneys,
   • urea accounts for approx. 90% of all bodily nitrogenous excretory products.
   • ornithine is synthesized from glucose; arginine is synthesized from ornithine by the urea cycle