Carbamyl phosphate-aspartate transcarbamylase

I. Carbamyl Phosphate-Aspartate Transcarbamylase (Aspartate Carb-... 

Animal and bacterial enzymes that utilize or synthesize carbamyl phosphate have activity with acetyl phosphate. Acyl phosphatase hydrolyzes both substrates, and maybe involved in the specific dynamic action of proteins. Ornithine and aspartic transcarbamylases also synthesize acetylornithine and acetyl aspartate. Finally, bacterial carbamate kinase and animal carbamyl phosphate synthetase utilize acetyl phosphate as well as carbamyl phosphate in the synthesis of adenosine triphosphate. The synthesis of acetyl phosphate and of formyl phosphate by carbamyl phosphate synthetases is described. The mechanism of carbon dioxide activation by animal carbamyl phosphate synthetase is reviewed on the basis of the findings concerning acetate and formate activation. 

Three-dimensional structure of E. coli aspartate transcarbamylase. Half of the native c6r6 molecule (see Fig. II-4) is shown. The catalytic (c) submit, which binds the substrates aspartate and carbamyl phosphate is shown in light shading. The regulatory (r) subunit, which binds the allosteric effectors CTP and ATP, is shown in dark shading. From Kantrowitz, E.R., et al. (1980). E. coli Aspartate Transcarbamylase. Part 11 Structure and Allosteric Interactions. Trends Biochem Sci 5 150 and Stryer, L. (1995). Biochemistry, 4th ed. New York Freeman, Figure 10-5, p. 240. Reprinted by permission. 

The utilization of ammonia resulting from the combination of carbamyl phosphate with aspartic acid, the initial reaction for the synthesis of the pyrimidine nucleotides, continues only as long as there is a requirement for them (Fig. 3). Regulation of this biosynthetic pathway is probably by way of feedback inhibition of aspartate transcarbamylase. The rat liver enzyme is inhibited by uridine, cytidine or thymidine or such derivatives as CMP, UTP, or TMP, all intermediates or products of this pathway (B8). This is not the only enzyme of the pathway which may be subject to feedback regulation. Dihydroorotase from rat liver is also inhibited by some pyrimidines and purines (B9). 

Figure 7. Interactions at the active site of aspartate transcarbamylase (ATCase). N-phosphonoacetyl-L-asparate (PALA) is a bisubstrate analog of the two natural substrates of ATCase, carbamyl phosphate and L-aspartate. PALA is shown bound in the active site of ATCase. Noncovalent interactions between PALA and side-chains of the protein are shown as dashed lines. Specific residues are indicated by their one letter abbreviation and by their position in the protein sequence (e.g., HI 34 = histidine at position 134). The active site is composed of residues from two separate polypeptide chains (denoted by primed and unprimed residue numbers). Note the complimentarity of the site and the ligand. The same interactions are used to align and catalyze the condensation of ATCase s natural substrates (Monaco et al., 1978).

Another form of spatial organization of metabolism that is often seen in eukaryotes but is less common in bacteria involves enzyme aggregates or multifunctional enzymes. An example is seen in S. cerevisiae where the first two reactions in pyrimidine nucleotide biosynthesis, the synthesis of carbamyl phosphate and the carbamylation of aspartate, are catalyzed by a single bifunctional protein (31). Both reactions are subject to feedback inhibition by UTP, in contrast to the situation inB. subtilis where aspartate transcarbamylase activity is not controlled. It is possible that an evolutionary advantage of the fusion of the genes... 

The intramitochondrial location of the arginine-specific carbamyl phosphate synthetase in N. crassa has the additional advantage of assuring separate pools of carbamyl phosphate for arginine and pyrimidine biosynthesis (41). Since this precludes the utilization of carbamyl phosphate produced in the course of arginine biosynthesis by aspartate transcarbamylase and of the pyrimidine-specific carbamyl phosphate by ornithine transcarbamylase, the control of these reac-... 

The first step in pyrimidine biosynthesis is the reaction catalyzed by aspartic transcarbamylase, a reaction in which the carbamyl group of carbamyl phosphate is transferred to aspartic acid to yield ureidosuccinic acid. In E. coli, the end products of the pyrimidine pathways, UTP and especially CTP, inhibit the transcarbamylase. Pardee and his associate, who discovered this important event, established that the site of action in the transcarbamylase molecule of the inhibitor is different from the site of action of the substrate. 

The overall capacity of pyrimidine nucleotide de novo synthesis appears to be higher in rat liver than in rat brain. This can also be concluded from the higher activities of carbamyl phosphate synthetase II and aspartate transcarbamylase in liver (2,4). The liver primarily depends on the de novo pathway for nucleotide synthesis. With liver slices pyrimidine nucleotides are predominantly derived from OA uridine is mainly catabolyzed to uracil and 3-alanine (16) in agreement with high activity of uridine phosphorylase. With brain slices uridine was superior to CO2 or OA in labelling RNA (8). This concords with the relatively high activity of uridine kinase. In vivo, however, cytidine appears to be a more important substrate for nucleotide synthesis (17), since uridine in predominantly catabolyzed by various tissues, including liver. 

While the studies of Boyland and Roller and Elion and co-workers, which were conducted in vivo, do suggest that urethane has a specificity for pyrimidine biosynthesis, Kaye could not demonstrate in vitro any significant inhibition by urethane of several enzymes involved in nucleic acid metabolism. Both urethane and its A -hydroxy metabolite bear a structural resemblance to carbamyl phosphate and carbamyl-L-aspartate. The enzyme aspartate transcarbamylase begins pyrimidine biosynthesis by catalyzing the formation of carbamyl-L-aspartate from carbamyl phosphate and l-aspartate. Giri and Bhide have reported that in vivo administration of urethane decreased aspartate transcarbamylase activity of lung tissue of adult male and (to a lesser extent) female mice no in vitro inhibition could be demonstrated. 

The first step of the biosynthesis of pyrimidine nucleotides is the irreversible carbamylation of L-aspartate by carbamyl-phosphate to form carbamylaspartate (catalyzed by the enzyme aspartate transcarbamylase). Next, carbamylaspartate is converted, by ring closure, to dihydro-orotic acid which, in turn, is reduced to orotic acid, catalyzed by the enzyme orotic acid dehydrogenase (OAD). Orotic acid (6-carboxyuracil) reacts with 5 -phosphoribosyl--1-pyrophosphate (PRPP) to form orotidine monophosphate (OMP). 

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