l-Amino acid oxidase of liver and kidney convert an amino acid to an α-imino acid that decomposes to an α-keto acid with release of ammonium ion (Figure 28–13). The reduced flavin is reoxidized by molecular oxygen, forming hydrogen peroxide (H2O2), which then is split to O2 and H2O by catalase, EC 188.8.131.52.
Oxidative deamination catalyzed by l-amino acid oxidase (l-α-amino acid:O2 oxidoreductase, EC 184.108.40.206. The α-imino acid, shown in brackets, is not a stable intermediate.
Ammonia Intoxication Is Life Threatening
The ammonia produced by enteric bacteria and absorbed into portal venous blood and the ammonia produced by tissues are rapidly removed from circulation by the liver and converted to urea. Thus, only traces (10-20 μg/dL) normally are present in peripheral blood. This is essential, since ammonia is toxic to the central nervous system. Should portal blood bypass the liver, systemic blood ammonia may attain toxic levels. This occurs in severely impaired hepatic function or the development of collateral links between the portal and systemic veins in cirrhosis. Symptoms of ammonia intoxication include tremor, slurred speech, blurred vision, coma, and ultimately death. Ammonia may be toxic to the brain in part because it reacts with α-ketoglutarate to form glutamate. The resulting depletion of levels of α-ketoglutarate then impairs function of the tricarboxylic acid (TCA) cycle in neurons.
Formation of glutamine is catalyzed by mitochondrial glutamine synthetase (Figure 28–14). Since amide bond synthesis is coupled to the hydrolysis of ATP to ADP and Pi, the reaction strongly favors glutamine synthesis. During catalysis, glutamate attacks the γ-phosphoryl group of ATP, forming γ-glutamyl phosphate and ADP. Following deprotonation of NH4+, NH3 attacks γ-glutamyl phosphate, and glutamine and Pi are released. In addition to providing glutamine to serve as a carrier of nitrogen, carbon and energy between organs (Figure 28–5), glutamine synthetase plays a major role in ammonia detoxification and acid-base homeostasis. A rare deficiency in neonate glutamine synthetase results in severe brain damage, multiorgan failure, and death.
There are two human isoforms of mitochondrial glutaminase, termed liver-type and renal-type glutaminase. Products of different genes, the glutaminases differ with respect to their structure, kinetics, and regulation. Hepatic glutaminase levels rise in response to high protein intake while renal kidney-type glutaminase increases in metabolic acidosis. Hydrolytic release of the amide nitrogen of glutamine as ammonia, catalyzed by glutaminase (Figure 28–15), strongly favors glutamate formation. An analogous reaction is catalyzed by l-asparaginase (EC 220.127.116.11). The concerted action of glutamine synthetase and glutaminase thus catalyzes the interconversion of free ammonium ion and glutamine.
The reaction catalyzed by glutaminase, EC 18.104.22.168. The reaction proceeds essentially irreversibly in the direction of glutamate and NH4+ formation. Note that the amide nitrogen, not the α-amino nitrogen, is removed.
Formation & Secretion of Ammonia Maintains Acid-Base Balance
Excretion into urine of ammonia produced by renal tubular cells facilitates cation conservation and regulation of acid-base balance. Ammonia production from intracellular renal amino acids, especially glutamine, increases in metabolic acidosis and decreases in metabolic alkalosis.
Urea is the Major End Product of Nitrogen Catabolism in Humans
Synthesis of 1 mol of urea requires 3 mol of ATP, 1 mol each of ammonium ion and of aspartate, and employs five enzymes (Figure 28–16). Of the six participating amino acids, N-acetylglutamate functions solely as an enzyme activator. The others serve as carriers of the atoms that ultimately become urea. The major metabolic role of ornithine, citrulline, and argininosuccinate in mammals is urea synthesis. Urea synthesis is a cyclic process. While ammonium ion, CO2, ATP, and aspartate are consumed, the ornithine consumed in reaction 2 is regenerated in reaction 5. There thus is no net loss or gain of ornithine, citrulline, argininosuccinate, or arginine. As indicated in Figure 28–16, some reactions of urea synthesis occur in the matrix of the mitochondrion, and other reactions in the cytosol.
Reactions and intermediates of urea biosynthesis. The nitrogen-containing groups that contribute to the formation of urea are shaded. Reactions
occur in the matrix of liver mitochondria and reactions
in liver cytosol. CO2
(as bicarbonate), ammonium ion, ornithine, and citrulline enter the mitochondrial matrix via specific carriers (see red dots) present in the inner membrane of liver mitochondria.
Carbamoyl Phosphate Synthetase I Initiates Urea Biosynthesis
Condensation of CO2, ammonia, and ATP to form carbamoyl phosphate is catalyzed by mitochondrial carbamoyl phosphate synthetase I (EC 22.214.171.124). A cytosolic form of this enzyme, carbamoyl phosphate synthetase II, uses glutamine rather than ammonia as the nitrogen donor and functions in pyrimidine biosynthesis (see Figure 33–9). The concerted action of glutamate dehydrogenase and carbamoyl phosphate synthetase I thus shuttles amino nitrogen into carbamoyl phosphate, a compound with high group transfer potential.
Carbamoyl phosphate synthetase I, the rate-limiting enzyme of the urea cycle, is active only in the presence of N-acetylglutamate, an allosteric activator that enhances the affinity of the synthetase for ATP. Synthesis of 1 mol of carbamoyl phosphate requires 2 mol of ATP. One ATP serves as the phosphoryl donor for formation of the mixed acid anhydride bond of carbamoyl phosphate. The second ATP provides the driving force for synthesis of the amide bond of carbamoyl phosphate. The other products are 2 mol of ADP and 1 mol of Pi (reaction 1, Figure 28–16). The reaction proceeds stepwise. Reaction of bicarbonate with ATP forms carbonyl phosphate and ADP. Ammonia then displaces ADP, forming carbamate and orthophosphate. Phosphorylation of carbamate by the second ATP then forms carbamoyl phosphate.
Carbamoyl Phosphate Plus Ornithine Forms Citrulline
l-Ornithine transcarbamoylase (EC 126.96.36.199) catalyzes transfer of the carbamoyl group of carbamoyl phosphate to ornithine, forming citrulline and orthophosphate (reaction 2, Figure 28–16). While the reaction occurs in the mitochondrial matrix, both the formation of ornithine and the subsequent metabolism of citrulline take place in the cytosol. Entry of ornithine into mitochondria and exodus of citrulline from mitochondria therefore involve mitochondrial inner membrane permeases (Figure 28–16).
Citrulline Plus Aspartate Forms Argininosuccinate
Argininosuccinate synthetase (EC 188.8.131.52) links aspartate and citrulline via the amino group of aspartate (reaction 3, Figure 28–16) and provides the second nitrogen of urea. The reaction requires ATP and involves intermediate formation of citrullyl-AMP. Subsequent displacement of AMP by aspartate then forms argininosuccinate.
Cleavage of Argininosuccinate Forms Arginine & Fumarate
Cleavage of argininosuccinate is catalyzed by argininosuccinate lyase (EC 184.108.40.206). The reaction proceeds with retention of all three nitrogens in arginine and release of the aspartate skeleton as fumarate (reaction 4, Figure 28–16). Subsequent addition of water to fumarate forms l-malate, whose subsequent NAD+-dependent oxidation forms oxaloacetate. These two reactions are analogous to reactions of the citric acid cycle, but are catalyzed by cytosolic fumarase and malate dehydrogenase. Transamination of oxaloacetate by glutamate aminotransferase then re-forms aspartate. The carbon skeleton of aspartate-fumarate thus acts as a carrier of the nitrogen of glutamate into a precursor of urea.
Cleavage of Arginine Releases Urea & Re-Forms Ornithine
Hydrolytic cleavage of the guanidino group of arginine, catalyzed by liver arginase (EC 220.127.116.11), releases urea (reaction 5, Figure 28–16). The other product, ornithine, reenters liver mitochondria and participates in additional rounds of urea synthesis. Ornithine and lysine are potent inhibitors of arginase, and compete with arginine. Arginine also serves as the precursor of the potent muscle relaxant nitric oxide (NO) in a Ca2+-dependent reaction catalyzed by NO synthetase.
Carbamoyl Phosphate Synthetase I Is the Pacemaker Enzyme of the Urea Cycle
The activity of carbamoyl phosphate synthetase I is determined by N-acetylglutamate, whose steady-state level is dictated by the balance between its rate of synthesis from acetyl-CoA and glutamate and its rate of hydrolysis to acetate and glutamate, reactions catalyzed by N-acetylglutamate synthetase (NAGS) and N-acetylglutamate deacylase (hydrolase), respectively.
Acetyl-CoA + l-glutamate → N-acetyl-l-glutamate + CoASH
N-acetyl-l-glutamate + H2O → l-glutamate + acetate
Major changes in diet can increase the concentrations of individual urea cycle enzymes 10- to 20-fold. For example, starvation elevates enzyme levels, presumably to cope with the increased production of ammonia that accompanies enhanced starvation-induced degradation of protein.