Chapter 23:  23.4 Ammonium Ion Is Converted Into Urea in Most Terrestrial Vertebrates

23.4.1. The Urea Cycle Begins with the Formation of Carbamoyl Phosphate

The urea cycle begins with the coupling of free NH₄⁺ with HCO₃^- to form carbamoyl phosphate. The synthesis of carbamoyl phosphate, though a simple molecule, is complex, comprising three steps, all catalyzed by carbamoyl phosphate synthetase.

The reaction begins with the phosphorylation of HCO₃^- to form carboxyphosphate, which then reacts with ammonium ion to form carbamic acid. Finally, a second molecule of ATP phosphorylates carbamic acid to carbamoyl phosphate. The structure and mechanism of the fascinating enzyme that catalyzes these reactions will be discussed in Chapter 25. The consumption of two molecules of ATP makes this synthesis of carbamoyl phosphate essentially irreversible. The mammalian enzyme requires N-acetyl-glutamate for activity, as will be discussed shortly.

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

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, 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 cytosol.

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 cleaves argininosuccinate 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.

23.4.2. The Urea Cycle Is Linked to the Citric Acid Cycle

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 links the urea cycle and the citric acid cycle (Figure 23.17). Fumarate is hydrated to malate, which is in turn oxidized to oxaloacetate. Oxaloacetate has several possible fates: (1) transamination to aspartate, (2) conversion into glucose by the gluconeogenic pathway, (3) condensation with acetyl CoA to form citrate, or (4) conversion into pyruvate.

23.4.3. The Evolution of the Urea Cycle

We have previously encountered carbamoyl phosphate as a sub- strate for aspartate transcarbamoylase, the enzyme that catalyzes the first step in pyrimidine biosynthesis (Section 10.1). Carbamoyl phosphate synthetase generates carbamoyl phosphate for both this pathway and the urea cycle. In mammals, two distinct carbamoyl phosphate synthetase isozymes are present. As discussed earlier, the mitochondrial enzyme uses NH₄⁺ as the nitrogen source, as is appropriate for its role in the urea cycle. In pyrimidine biosynthesis, carbamoyl phosphate synthetase differs in two important ways (Section 25.1.1). First, this enzyme utilizes glutamine as a nitrogen source. The side chain amide is hydrolyzed within one domain of the enzyme and the ammonia generated moves through a tunnel in the enzyme to react with carboxyphosphate. Second, this enzyme is part of a large polypeptide called CAD that comprises three distinct enzymes: carbamoyl phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase, all of which catalyze 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. N-Acetylglutamate is synthesized only if free amino acids are present, an indication that any ammonia generated must be disposed of. A catalytic site in one isozyme has been adapted to act as an allosteric site in another isozyme having a different physiological role.

What about the other enzymes in the urea cycle? Ornithine transcarbamoylase is homologous to aspartate transcarbamoylase and the structures of their catalytic subunits are quite similar (Figure 23.18). 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 25.2.3).

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.

23.4.4. Inherited Defects of the Urea Cycle Cause Hyperammonemia and Can Lead to Brain Damage

The synthesis of urea in the liver is the major route of removal of NH₄⁺. 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. Why are high levels of NH₄⁺ toxic? The answer to this question is not yet known. One possibility is that elevated levels of glutamine, formed from NH₄⁺ and glutamate (Section 23.3.5), produce osmotic effects that lead directly to brain swelling.

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 23.19). This urea-cycle intermediate condenses with aspartate to yield argininosuccinate, which is then excreted. Note that two nitrogen atoms—one from carbamoyl phosphate and another 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 these two amino acids, which 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 (Figure 23.20). Likewise, phenylacetate is activated to phenylacetyl CoA, which reacts with glutamine to form phenylacetylglutamine. These conjugates substitute for urea in the disposal of nitrogen. Thus, latent biochemical pathways can be activated to partly bypass a genetic defect.

23.4.5. Urea Is Not the Only Means of Disposing of Excess Nitrogen

As discussed 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, which secrete nitrogen as the purine uric acid, require little water. Disposal of excess nitrogen as uric acid is especially valuable in animals, such as birds, that produce eggs having impermeable membranes that accumulate waste products. The pathway for nitrogen excretion developed in the course of evolution clearly depends on the habitat of the organism.