N-carbamoyl-L-aspartate

ACTase catalyzes the transfer of a carbamoyl residue from carbamoyl phosphate to the amino group of L-aspartate. The N-carbamoyl L-aspartate formed in this way already contains all of the atoms of the later pyrimidine ring (see p. 188). The ACTase of the bacterium Escherichia coli is inhibited by cytidine triphosphate (CTP), an end product of the anabolic metabolism of pyrimidines, and is activated by the precursor ATP. 

Figure 7.6 (a) The first step in the biosynthesis of pyrimidines, (b) The proposed transition state for the carbamoyl phosphate/aspartic acid stage in pyrimidine synthesis, (c) The structure of sodium N-phosphonoacetyl-L-aspartate (PALA)... 

The natural function of the carboxymethylhydantoinase (E. C. 3.5.2.2) is postulated to be the hydrolysis of 5-carboxymethylhydantoin, which is described to be the product of a non-enzymatic cyclization of N-carbamoyl-i-aspartic acid123, 241 and to occur as a side-product in the metabolism of the pyrimidine nucleotide dihydroorotic acid1251. This enzyme often occurs in combination with a ureidosuccinase (E.C. 3.5.1.7)[2S1, which catalyzes the cleavage of the resulting N-carbamoyl aspartic acid to L-aspartic acid (see Fig. 12.4-5). L-5-Carboxymethylhydantoin was first isolated after incubating orotic acid, a six-membered cyclic amide, with crude cell extracts of the anaerobic bacterium Clostridium oroticum125, 261. 

A particularly intuitive application of this concept may be the experimental anticancer drug N-(phosphonoacetyl)-L-aspartate (PALA). The first step in the de novo biosynthesis of the pyrimidine nucleotide formation in the cell involves the condensation of carbamoyl phosphate with L-aspartic acid catalyzed by the enzyme aspartate transcarbamylase (Eq. 2.14).2 One can postulate a transition state, as shown in Eq. 2.14. 

Figure 8.19. Sequence reactions from aspartic acid (AA) and carbamoyl phosphate (CP) to the end product, cytidine triphosphate (CTP). The first reaction is catalyzed by ATCase. The intermediary compounds are N-carbamoyl aspartic acid (N-CAA), L-dihydroorotic acid (L-DHOA), orotic acid (OA), orotidine 5 -phosphate (0-5 -P), uridine 5 -phosphate (U-5 -P), uridine diphosphate (UDP), and uridine triphosphate (UTP).

Figure 10.6. PALA, a Bisubstrate Analog. (Top) Nucleophilic attack by the amino group of aspartate on the carbonyl carbon atom of carbamoyl phosphate generates an intermediate on the pathway to the formation of N-carbamoylaspartate. (Bottom) A-(Phosphonacetyl)-l-aspartate (PALA) is an analog of the reaction intermediate and a potent competitive inhibitor of aspartate transcarbamoylase.

Reaction of aspartic acid (14) with carbamoyl phosphoric acid (17) in the presence of the allosteric enzyme aspartate carbamoyltransferase (aspartate transcar-bamoylase) gives N-carbamoyl aspartic acid (18), which is cyclised to L-dihy-droorotic acid (19) by dihydroorotase. Oxidation of L-dihydroorotic acid by flavoprotein, orotate reductase gives orotic acid (20), which reacts with 5-phosphori-bosy 1-1-pyrophosphate (PRPP) in the presence of orotate phosphoribosyl transferase to form orotidine 5 -monophosphate (OMP, 21). Decarboxylation of OMP by orotid-ine 5 -phosphate decarboxylase yields uridine 5 -monophosphate (UMP, 22), which acts as precursor for the cytidine nucleotides (CTP) (Chart 6). 

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