Urea Is The Major End Product Of Nitrogen Catabolism In Humans

Synthesis of 1 mol of urea requires 3 mol of ATP plus 1 mol each of ammonium ion and of the a-amino nitrogen of aspartate. Five enzymes catalyze the numbered

L-Glutamine

GLUTAMINASE

L-Glutamate

Figure 29-8. The glutaminase reaction proceeds essentially irreversibly in the direction of glutamate and NH4+ formation. Note that the amide nitrogen, not the a-amino nitrogen, is removed.

reactions of Figure 29-9. Of the six participating amino acids, ^-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 argininosucci-nate in mammals is urea synthesis. Urea synthesis is a cyclic process. Since the ornithine consumed in reaction 2 is regenerated in reaction 5, there is no net loss or gain of ornithine, citrulline, argininosuccinate, or arginine. Ammonium ion, CO2, ATP, and aspartate are, however, consumed. Some reactions of urea synthesis occur in the matrix of the mitochondrion, other reactions in the cytosol (Figure 29-9).

L-Glutamate

GLUTAMINE SYNTHASE

H2NN

L-Glutamine

Figure 29-7. The glutamine synthase reaction strongly favors glutamine synthesis.

Carbamoyl Phosphate Synthase I Initiates Urea Biosynthesis

Condensation of CO2, ammonia, and ATP to form carbamoyl phosphate is catalyzed by mitochondrial carbamoyl phosphate synthase I (reaction 1, Figure 29-9). A cytosolic form of this enzyme, carbamoyl phosphate synthase II, uses glutamine rather than ammonia as the nitrogen donor and functions in pyrimi-dine biosynthesis (see Chapter 34). Carbamoyl phosphate synthase I, the rate-limiting enzyme of the urea cycle, is active only in the presence of its allosteric activator A-acetylglutamate, which enhances the affinity of the synthase for ATP. Formation of carbamoyl phosphate requires 2 mol of ATP, one of which serves as a phosphate donor. Conversion of the second ATP to AMP and pyrophosphate, coupled to the hydrolysis of pyrophosphate to orthophosphate, provides the driving

AvjvA

CH2-NH

AvjvA

CH2-NH

HC-COO-

ooc-Ch

Fumarate

CH2-NH

HC-COO-

ooc-Ch

Fumarate

CH2-NH

L-Citrulline

COO-

NH COO-

ch2-Nh Ch2

CH2 COO-

Argininosuccinate

COO-

COO-H2N-C-H

COO-L-Aspartate

Figure 29-9. Reactions and intermediates of urea biosynthesis. The nitrogen-containing groups that contribute to the formation of urea are shaded. Reactions ©and © occur in the matrix of liver mitochondria and reactions ©, ©, and © in liver cytosol. CO2 (as bicarbonate), ammonium ion, ornithine, and cit-rulline enter the mitochondrial matrix via specific carriers (see heavy dots) present in the inner membrane of liver mitochondria.

force for synthesis of the amide bond and the mixed acid anhydride bond of carbamoyl phosphate. The concerted action of GDH and carbamoyl phosphate synthase I thus shuttles nitrogen into carbamoyl phosphate, a compound with high group transfer potential. 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 catalyzes transfer of the carbamoyl group of carbamoyl phosphate to ornithine, forming citrulline and orthophosphate (reaction 2, Figure 29-9). 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 transport systems (Figure 29-9).

Citrulline Plus Aspartate Forms Argininosuccinate

Argininosuccinate synthase links aspartate and citrulline via the amino group of aspartate (reaction 3, Figure 29-9) 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 citrulline.

Cleavage of Argininosuccinate Forms Arginine & Fumarate

Cleavage of argininosuccinate, catalyzed by argini-nosuccinase, proceeds with retention of nitrogen in arginine and release of the aspartate skeleton as fu-marate (reaction 4, Figure 29-9). Addition of water to fumarate forms L-malate, and subsequent NAD+-dependent oxidation of malate forms oxaloacetate. These two reactions are analogous to reactions of the citric acid cycle (see Figure 16-3) 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, releases urea (reaction 5, Figure 29-9). The other product, ornithine, reenters liver mitochondria for additional rounds of urea synthesis. Ornithine and lysine are potent inhibitors of arginase, competitive with arginine. Arginine also serves as the precursor of the potent muscle relaxant nitric oxide (NO) in a Ca2+-dependent reaction catalyzed by NO synthase (see Figure 49-15).

Carbamoyl Phosphate Synthase I Is the Pacemaker Enzyme of the Urea Cycle

The activity of carbamoyl phosphate synthase I is determined by A-acetylglutamate, whose steady-state level is dictated by its rate of synthesis from acetyl-CoA and glutamate and its rate of hydrolysis to acetate and glutamate. These reactions are catalyzed by A-acetylglu-tamate synthase and A-acetylglutamate hydrolase, respectively. Major changes in diet can increase the concentrations of individual urea cycle enzymes 10-fold to 20-fold. Starvation, for example, elevates enzyme levels, presumably to cope with the increased production of ammonia that accompanies enhanced protein degradation.

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Diabetes 2

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