Argininosuccinate, synthesis

The six reactions listed in Table 5-IV are similar in that (1) protonated ammonia or a carrier of protonated ammonia is involved (2) all are reversible (3) no isotope exchange or reaction occurs in either direction unless all three reactants are present (4) all require ATP or GTP (5) 0 from phosphate is transferred to the amino acceptor. (The case of argininosuccinate synthesis is different in that pyrophosphate is formed, and from citrulline is found in adenylate rather than in pyrophosphate.)... 

Two ATP molecules are used in the carbamoyl phosphate synthase reaction and one in argininosuccinate synthesis. Thus urea synthesis may be regarded as an energetically expensive process, although ATP is synthesized during the mitochondrial oxidation of the NADH produced by the oxidative deamination of the amino acids. 

Both the ammonia and the aspartate required for argininosuccinate synthesis are derived from glutamate. The ammonia is formed by the glutamate dehydrogenase reaction while aspartate is formed by the transamination of glutamate with oxaloacetate via the glutamate-oxaloacetate transaminase (GOT) reaction. 

Patients who survive the neonatal period can be maintained with a low-protein diet and sodium benzoate. A useful therapeutic adjunct for citrullinemia and argininosuccinic aciduria is dietary arginine supplementation, which enhances the ability to eliminate nitrogen as either citrulline or argininosuccinate. Maintaining normal arginine levels also facilitates protein synthesis. 

If we consider the urea cycle in isolation, we see that the synthesis of one molecule of urea requires four high-energy phosphate groups (Fig. 18-10). Two ATP molecules are required to make carbamoyl phosphate, and one ATP to make argininosuccinate—the latter ATP undergoing a pyrophosphate cleavage to AMP and PPj, which is hydrolyzed to two Pj. The overall equation of the urea cycle is... 

Argininosuccinic acidemia <1.5 Urea synthesis Argininosuccinase Vomiting convulsions... 

Synthesis of argininosuccinate Citrulline condenses with aspar tate to form argininosuccinate. The a-amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea. The formation of argininosuccinate is driven by the cleav age of ATP to AMP and pyrophosphate (PPi). This is the third and final molecule of ATP consumed in the formation of urea. 

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. 

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). 

Arginine and fumarate are produced from argininosuccinate by the cytosolic enzyme argininosuccinate lyase. In the final step of the cycle arginase cleaves urea from aspartate, regenerating cytosolic ornithine, which can be transported to the mitochondrial matrix for another round of urea synthesis. 

Fig. 3. Uptake of ammonia in pyrimidine synthesis and breakdown. Compounds found in excess in inherited disorders of urea synthesis are enclosed in rectangles. ASA, argininosuccinate.

Arginine or citrulline in concentration in excess of the optimal can inhibit both argininosuccinate synthetase or argininosuccinate lyase in tissue cultures (S6). Whether this is applicable to the conditions of synthesis of the urea in liver is uncertain. a-Methylaspartic acid also specifically inhibits argininosuccinate synthetase (B7, C4, SIO). 

Protein Intake and Urea Excretion. Levin et al. (L7) were the first to show that urea excretion and therefore presumably urea synthesis was increased with increased protein intake in argininosuccinic aciduria. They showed in their patient that an increase of 2.5 times in the protein intake resulted in a 4- or 5-fold increase in urea output. From the results of a feeding trial, in which the infant was given a casein hydrolyzate from which most of the arginine had been removed, they concluded from the small amount of arginine present and the relatively high amount of urea excreted daily, that most of the urea was derived from a urea cycle, presumably in the liver. Conversely, reduction of protein intake resulted in a marked decrease in the output of both urea and argininosuccinic acid. 

Cedrangolo et al (C5) and De Lorenzo (Dl) also postulated an alternative pathway on the basis of their experimental results on rats. The animals were injected with a-methyl aspartate, a specific inhibitor for argininosuccinate synthetase. No effect on urea excretion was observed, but there was complete inhibition of urea synthesis from citrulline in liver homogenates prepared from injected animals, as well as increased susceptibility to ammonia intoxication. However, this inhibition was not confirmed by Crokaert and Baroen (C15, C16, C17), although they did confirm the lack of any effect on urea excretion. The experimental basis for this suggestion is therefore in doubt. 

The reaction is driven forward by hydrolysis of pyrophosphate to inorganic phosphate. Argininosuccinate formation is considered as the rate-limiting step for urea synthesis. This reaction incorporates the second nitrogen atom of the urea molecule donated by aspartate. 

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