Some of the NH₄⁺ formed in the breakdown of amino acids is consumed in the biosynthesis of nitrogen compounds. In most terrestrial vertebrates, the excess NH₄⁺ is converted into urea and then excreted. Such organisms are referred to as ureotelic.

In terrestrial vertebrates, urea is synthesized by the urea cycle (Figure 23.15). One of the nitrogen atoms of urea is transferred from an amino acid, aspartate. The other nitrogen atom is derived directly from free NH₄⁺, and the carbon atom comes from HCO₃⁻ (derived by the hydration of CO₂; Section 9.2).

The urea cycle begins with the coupling of free NH₃ with HCO₃⁻ to form carbamoyl phosphate, the committed reaction of the urea cycle, which is catalyzed by carbamoyl phosphate synthetase I. Carbamoyl phosphate is a simple molecule, but its synthesis is complex, requiring three steps.

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Note that NH₃, because it is a strong base, normally exists as NH₄⁺ in aqueous solution. However, carbamoyl phosphate synthetase uses only NH₃ as a substrate. The reaction begins with the phosphorylation of HCO₃⁻ to form carboxyphosphate, which then reacts with NH₃ to form carbamic acid. Finally, a second molecule of ATP phosphorylates carbamic acid to form carbamoyl phosphate. The structure and mechanism of the enzyme that catalyzes these reactions will be presented in Chapter 25. The consumption of two molecules of ATP makes this synthesis of carbamoyl phosphate essentially irreversible.

Carbamoyl phosphate synthetase is regulated both allosterically and by covalent modification so that it is maximally active when amino acids are being metabolized for fuel use. The allosteric regulator N-acetylglutamate (NAG) is required for synthetase activity. This molecule is synthesized by N-acetylglutamate synthase.

N-acetylglutamate synthase is itself activated by arginine. Thus, NAG is synthesized when amino acids, as represented by arginine and glutamate, are readily available, and carbamoyl phosphate synthetase is then activated to process the generated ammonia. When ammonia is not being generated, the synthetase is inhibited by acetylation. A rise in the mitochondrial NAD⁺, indicative of an energy-poor state, stimulates a deacetylase that removes the acetyl group, activating the synthetase and readying the enzyme for processing ammonia from protein degradation. The control of the acetylation of the synthetase is not yet clear.

The carbamoyl group of carbamoyl phosphate has a high transfer potential because of its anhydride linkage. The carbamoyl group is transferred to ornithine to form citrulline, in a reaction catalyzed by ornithine transcarbamoylase.

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Ornithine and citrulline are amino acids, but they are not used as building blocks of proteins. The formation of NH₄⁺ by glutamate dehydrogenase, its incorporation into carbamoyl phosphate as NH₃, and the subsequent synthesis of citrulline take place in the mitochondrial matrix. In contrast, the next three reactions of the urea cycle, which lead to the formation of urea, take place in the cytoplasm.

Citrulline is transported to the cytoplasm, where it condenses with aspartate, the donor of the second amino group of urea. This synthesis of argininosuccinate, catalyzed by argininosuccinate synthetase, is driven by the cleavage of ATP into AMP and pyrophosphate and by the subsequent hydrolysis of pyrophosphate.

Argininosuccinase (also called argininosuccinate lyase) cleaves arginino-succinate into arginine and fumarate. Thus, the carbon skeleton of aspartate is preserved in the form of fumarate.

Finally, arginine is hydrolyzed to generate urea and ornithine in a reaction catalyzed by arginase. Ornithine is then transported back into the mitochondrion to begin another cycle. The urea is excreted. Indeed, human beings excrete about 10 kg (22 pounds) of urea per year.

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The stoichiometry of urea synthesis is

Pyrophosphate is rapidly hydrolyzed, and so the equivalent of four molecules of ATP are consumed in these reactions to synthesize one molecule of urea. The synthesis of fumarate by the urea cycle is important because it is a precursor for glucose synthesis (Figure 23.16). Fumarate is hydrated to malate, which is in turn oxidized to oxaloacetate. Oxaloacetate can be converted into glucose by gluconeogenesis or transaminated to aspartate.

Carbamoyl phosphate synthetase generates carbamoyl phosphate for both the urea cycle and the first step in pyrimidine biosynthesis (Section 25.1). In mammals, two distinct isozymes of the enzyme are present. Carbamoyl phosphate synthetase II, used in pyrimidine biosynthesis, differs in two important ways from its urea-cycle counterpart. First, this enzyme utilizes glutamine as a nitrogen source rather than NH₃. The sidechain amide of glutamine is hydrolyzed within one domain of the enzyme, and the ammonia generated moves through a tunnel in the enzyme to a second active site, where it reacts with carboxyphosphate. Second, this enzyme is part of a large complex that catalyzes several steps in pyrimidine biosynthesis (Section 25.1). Interestingly, the domain in which glutamine hydrolysis takes place is largely preserved in the urea-cycle enzyme, although that domain is catalytically inactive. This site binds N-acetylglutamate, an allosteric activator of the enzyme. A catalytic site in one isozyme has been adapted to act as an allosteric site in another isozyme having a different physiological role.

Can we find homologs for the other enzymes in the urea cycle? Ornithine transcarbamoylase is homologous to aspartate transcarbamoylase, which catalyzes the first step in pyrimidine biosynthesis, and the structures of their catalytic subunits are quite similar (Figure 23.17). Thus, two consecutive steps in the pyrimidine biosynthetic pathway were adapted for urea synthesis. The next step in the urea cycle is the addition of aspartate to citrulline to form argininosuccinate, and the subsequent step is the removal of fumarate. These two steps together accomplish the net addition of an amino group to citrulline to form arginine. Remarkably, these steps are analogous to two consecutive steps in the purine biosynthetic pathway (Section 25.2).

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The enzymes that catalyze these steps are homologous to argininosuccinate synthetase and argininosuccinase, respectively. Thus, four of the five enzymes in the urea cycle were adapted from enzymes taking part in nucleotide biosynthesis. The remaining enzyme, arginase, appears to be an ancient enzyme found in all domains of life.

The synthesis of urea in the liver is the major route for the removal of NH₄⁺. Urea cycle disorders occur with a prevalence of about 1 in 15,000. A blockage of carbamoyl phosphate synthesis or of any of the four steps of the urea cycle has devastating consequences because there is no alternative pathway for the synthesis of urea. All defects in the urea cycle lead to an elevated level of NH₄⁺ in the blood (hyperammonemia). Some of these genetic defects become evident a day or two after birth, when the afflicted infant becomes lethargic and vomits periodically. Coma and irreversible brain damage may soon follow, a condition called hepatic encephalopathy. Why are high levels of NH₄⁺ toxic? The answer to this question is not yet known. Recent work, however, suggests that NH₄⁺ may inappropriately activate a sodium-potassium-chloride cotransporter. This activation disrupts the osmotic balance of the nerve cell, causing swelling that damages the cell and results in neurological disorders.

Ingenious strategies for coping with deficiencies in urea synthesis have been devised on the basis of a thorough understanding of the underlying biochemistry. Consider, for example, argininosuccinase deficiency. This defect can be partly bypassed by providing a surplus of arginine in the diet and restricting the total protein intake. In the liver, arginine is split into urea and ornithine, which then reacts with carbamoyl phosphate to form citrulline (Figure 23.18). This urea-cycle intermediate condenses with aspartate to yield argininosuccinate, which is then excreted. Note that two nitrogen atoms—one from carbamoyl phosphate and the other from aspartate—are eliminated from the body per molecule of arginine provided in the diet. In essence, argininosuccinate substitutes for urea in carrying nitrogen out of the body.

The treatment of carbamoyl phosphate synthetase deficiency or ornithine transcarbamoylase deficiency illustrates a different strategy for circumventing a metabolic block. Citrulline and argininosuccinate cannot be used to dispose of nitrogen atoms because their formation is impaired. Under these conditions, excess nitrogen accumulates in glycine and glutamine. The challenge then is to rid the body of the nitrogen accumulating in these two amino acids. That goal is accomplished by supplementing a protein-restricted diet with large amounts of benzoate and phenylacetate. Benzoate is activated to benzoyl CoA, which reacts with glycine to form hippurate, which is excreted (Figure 23.19). Likewise, phenylacetate is activated to phenylacetyl CoA, which reacts with glutamine to form phenylacetylglutamine, which is also excreted. These conjugates substitute for urea in the disposal of nitrogen. Thus, latent biochemical pathways can be activated to partly bypass a genetic defect.

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As stated earlier, most terrestrial vertebrates are ureotelic; they excrete excess nitrogen as urea. However, urea is not the only excretable form of nitrogen. Ammoniotelic organisms, such as aquatic vertebrates and invertebrates, release nitrogen as NH₄⁺ and rely on the aqueous environment to dilute this toxic substance. Interestingly, lungfish, which are normally ammoniotelic, become ureotelic in time of drought, when they live out of the water.

Both ureotelic and ammoniotelic organisms depend on sufficient water, to varying degrees, for nitrogen excretion. In contrast, uricotelic organisms, such as birds and reptiles, secrete nitrogen as the purine uric acid. Uric acid is secreted as an almost solid slurry requiring little water. The secretion of uric acid also has the advantage of removing four atoms of nitrogen per molecule. The pathway for nitrogen excretion developed in the course of evolution clearly depends on the habitat of the organism.