The synthesis of urea involves a series of reactions. The initial two reactions occur in the mitochondrial matrix, while the remaining enzymes are located in the cytosol. Gluconeogenesis and heme synthesis also involve both the mitochondrial matrix and the cytosol. Carbamoyl phosphate is formed through the action of carbamoyl phosphate synthetase I (CPS I), which requires the cleavage of two molecules of ATP. Ammonia, derived from the oxidative deamination of glutamate by mitochondrial GDH, is incorporated into carbamoyl phosphate and eventually becomes one of the nitrogen atoms in urea. N-acetylglutamate (NAG) acts as a positive allosteric activator for CPS I. In contrast, carbamoyl phosphate synthetase II is involved in pyrimidine biosynthesis and occurs in the cytosol, utilizing glutamine as the nitrogen source. The carbamoyl portion of carbamoyl phosphate is transferred to ornithine by ornithine transcarbamylase (OTC), resulting in the release of phosphate. The product of this reaction, citrulline, is transported to the cytosol. Ornithine and citrulline move across the inner mitochondrial membrane via an antiporter. These basic amino acids are not incorporated into cellular proteins. Ornithine is regenerated in each cycle of the urea cycle, similar to how oxaloacetate is regenerated in the tricarboxylic acid (TCA) cycle. Argininosuccinate formation occurs through the combination of citrulline and aspartate by argininosuccinate synthetase. The α-amino group of aspartate provides the second nitrogen incorporated into urea. The formation of argininosuccinate is driven by the cleavage of ATP to adenosine monophosphate and pyrophosphate, representing the third molecule of ATP consumed in urea formation. Argininosuccinate is then cleaved by argininosuccinate lyase, yielding arginine and fumarate. Arginine serves as the immediate precursor of urea, while fumarate is hydrated to malate, connecting with various metabolic pathways. Malate can be oxidized to oxaloacetate by malate dehydrogenase, which can then be transaminated to aspartate and enter the urea cycle. Alternatively, malate can be transported into mitochondria via the malate-aspartate shuttle, reenter the TCA cycle, and be oxidized to oxaloacetate for gluconeogenesis. Malate oxidation also generates NADH for oxidative phosphorylation, reducing the energy cost of the urea cycle.
A. Reactions
The first two reactions leading to the synthesis of urea occur in the mitochondrial matrix, whereas the remaining cycle enzymes are located in the cytosol (Fig. 19.14). [Note: Gluconeogenesis (see p. 117) and heme synthesis (see p. 278) also involve both the mitochondrial matrix and the cytosol.]
Pi = inorganic phosphate; NAD(H) = nicotinamide adenine dinucleotide; MD = malate dehydrogenase.
1.
Carbamoyl phosphate formation: Formation of carbamoyl phosphate by carbamoyl phosphate synthetase I (CPS I) is driven by cleavage of two molecules of ATP. Ammonia incorporated into carbamoyl phosphate is provided primarily by the oxidative deamination of glutamate by mitochondrial GDH (see Fig. 19.11). Ultimately, the nitrogen atom derived from this ammonia becomes one of the nitrogens of urea. CPS I requires N-acetylglutamate (NAG) as a positive allosteric activator (see Fig. 19.14). [Note: Carbamoyl phosphate synthetase II participates in the biosynthesis of pyrimidines (see p. 302). It does not require NAG, uses glutamine as the nitrogen source, and occurs in the cytosol.] 2.
Citrulline formation: The carbamoyl portion of carbamoyl phosphate is transferred to ornithine by ornithine transcarbamylase (OTC) as the phosphate is released as inorganic phosphate. The reaction product, citrulline, is transported to the cytosol. [Note: Ornithine and citrulline move across the inner mitochondrial membrane via an antiporter. These basic amino acids are not incorporated into cellular proteins because there are no codons for them (see p. 447).] Ornithine is regenerated with each turn of the urea cycle, much in the same way that oxaloacetate is regenerated by the reactions of the tricarboxylic acid (TCA) cycle (see p. 109).
3.
Argininosuccinate formation: Argininosuccinate synthetase combines citrulline with aspartate to form argininosuccinate. The α-amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea. The formation of argininosuccinate is driven by the cleavage of ATP to adenosine monophosphate and pyrophosphate. This is the third and final molecule of ATP consumed in the formation of urea.
4.
Argininosuccinate cleavage: Argininosuccinate is cleaved by argininosuccinate lyase to yield arginine and fumarate. The arginine serves as the immediate precursor of urea. The fumarate is hydrated to malate, providing a link with several metabolic pathways. Malate can be oxidized by malate dehydrogenase to oxaloacetate, which can be transaminated to aspartate (see Fig. 19.8) and enter the urea cycle (see Fig. 19.14). Alternatively, malate can be transported into mitochondria via the malate–aspartate shuttle (see p. 80), reenter the TCA cycle, and get oxidized to oxaloacetate, which can be used for gluconeogenesis (see p. 120). [Note: Malate oxidation generates NADH for oxidative phosphorylation (see p. 77), thereby reducing the energy cost of the urea cycle.] 5.