Five enzymatic reactions take place in the urea cycle, the first two of which take place in mitochondria, the other three in the cytosol.
Firstly carbamoyl phosphate synthesase, this is not technically part of the urea cycle, it catalyses the condensation and activation of ammonia from the deamination of glutamate by glutamate dehydrogenase, and CO2 (in the form of bicarbonate, HCO3-) to form carbamoyl phosphate. The hydrolysis of two ATP molecules makes this reaction essentially irreversible.
The second reaction also occurs in the mitochondria and involves the transfer of the carbamoyl group from carbamoyl phosphate to ornithine by ornithine transcarbamoylas. This reaction forms the second non standard amino acid – citrulline, this then has to be transported to the cytosol where the remaining three reactions take place.
Third reaction involves citrulline being condensed with aspartate, the source of the second nitrogen atom in urea, by the enzyme argininosuccinate synthetase to form argninosuccinate. This reaction is driven by the hydrolysis of ATP to AMP and PPi, with subsequent hydrolysis of the pyrophosphate. Thus both of the high energy bonds in ATP are ultimately cleaved.
Fourthly argninosuccinase then removes the carbon skeleton of aspartate from argninosuccinate in the form of fumarate, leaving the nitrogen atom on the other product arginine. As the urea cycle also produces arginine, this amino acid is classified as non-essential in ureotolic organisms.
Fifthly and finally urea is formed from arginine by the action of arginase with the regeneration of ornithine. The orninthine is then transported back into the mitochondrion ready to be combined with another molecule of carbamoyl phosphate.
The synthesis of fumarate by argininosuccinase in the fourth step of the cycle links the urea cycle to the citric acid cycle. Fumarate enters the mitochondria, where the combined activities of fumarase and malate dehydrogenase transform fumarate into oxaloacetate. Aspartate, which acts as a nitrogen donor in the cytosol, is formed from oxaloacetate by transamination from glutamate; the other product of this transamination is α-ketoglutarate, another intermediate of the citric acid cycle. Because the reactions or the urea and citric acid cycle are inextricably intertwined, together they have been called the ‘krebs bicycle’
The activity of the urea cycle is regulated. The flux of nitrogen throught the urea cycle varies with the composition of the diet. When the diet is primarily protein, the use of carbon skeletons of amino acids for fuels results in the production of much urea from excess amino groups. During severe starvation, when breakdown of muscle protein supplies much of the metabolic fuel the urea production also increases substantially, for the same reason.
These changes in demand for urea cycle activity are met in the long term by regulation of the rate of synthesis of the urea cycle enzymes and carbamoyl phosphate synthetase I in the liver. All five enzymes are synthesised at higher rates during starvation or in animals on very high proteins diets than in well fed animals on diets containing primarily carbohydrates and fats. Animals on protein free diets produce even lower levels of urea cycle enzymes. On a shorter scale , allosteric regulation of at least one key enzyme is involved in adjusting flux through the cycle. The first enzyme in the pathway, carbamoyl phosphate synthetase I is allosterically activiated by N-acetylglutamate, which is synthesised from acetyl CoA and glutamate. N-acetylglutamate synthase is in turn activated by arginine, a urea cycle intermediate that accumulates when urea production is too slow to accommodate the ammonia produced by amino acid catabolism
In conclusion ureotelic organisms need to convert ammonia to nitrogen, in order to reduce toxicity levels in the body. This is done by the urea cycle, using three non-standard amino acids ammonia is converted to urea via 5 enzymatic reactions, these reactions are closely intertwined with the citric acid cycle.
