Ammonium Ion Is Converted Into Urea in Most Terrestrial Vertebrates

II. Transducing and Storing Energy
23. Protein Turnover and Amino Acid Catabolism
23.3. The First Step in Amino Acid Degradation Is the Removal of Nitrogen
Figure 23.15. The Alanine Cycle. Glutamate in muscle is transaminated to alanine, which is released into the
bloodstream. In the liver, alanine is taken up and converted into pyruvate for subsequent metabolism.
II. Transducing and Storing Energy
23. Protein Turnover and Amino Acid Catabolism
23.4. Ammonium Ion Is Converted Into Urea in Most Terrestrial Vertebrates
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 23.16). The urea cycle, proposed by Hans Krebs
and Kurt Henseleit in 1932, was the first cyclic metabolic pathway to be discovered. One of the nitrogen atoms of the
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 hydration of CO2; see Section 9.2).
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 NH
+
4
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 NH
+
4
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.
II. Transducing and Storing Energy
23. Protein Turnover and Amino Acid Catabolism
23.4. Ammonium Ion Is Converted Into Urea in Most Terrestrial Vertebrates
Figure 23.16. The Urea Cycle.
II. Transducing and Storing Energy
23. Protein Turnover and Amino Acid Catabolism
23.4. Ammonium Ion Is Converted Into Urea in Most Terrestrial Vertebrates
Figure 23.17. Metabolic Integration of Nitrogen Metabolism. The urea cycle, the citric acid cycle, and the
transamination of oxaloacetate are linked by fumarate and aspartate.
II. Transducing and Storing Energy
23. Protein Turnover and Amino Acid Catabolism
23.4. Ammonium Ion Is Converted Into Urea in Most Terrestrial Vertebrates
Figure 23.18. Homologous Enzymes. The structure of the catalytic subunit of ornithine transcarbamoylase (blue) is
quite similar to that of the catalytic subunit of aspartate transcarbamoylase (red), indicating that these two
enzymes are homologs.
II. Transducing and Storing Energy
23. Protein Turnover and Amino Acid Catabolism
23.4. Ammonium Ion Is Converted Into Urea in Most Terrestrial Vertebrates
Figure 23.19. Treatment of Argininosuccinase Deficiency. Argininosuccinase deficiency can be managed by
supplementing the diet with arginine. Nitrogen is excreted in the form of argininosuccinate.
II. Transducing and Storing Energy
23. Protein Turnover and Amino Acid Catabolism
23.4. Ammonium Ion Is Converted Into Urea in Most Terrestrial Vertebrates
Figure 23.20. Treatment of Carbamoyl Phosphate Synthetase and Ornithine Transcarbamoylase Deficiencies.
Both deficiencies can be treated by supplementing the diet with benzoate and phenylacetate. Nitrogen is excreted in the
form of hippurate and phenylacetylglutamine.