N-carbamoyl aspartate

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).

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). 

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. 

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)... 

Aspartate transcarbamoylase (aspartate carbamoyltransferase ATCase), a key enzyme in pyrimidine biosynthesis (see Topic FI), provides a good example of allosteric regulation. ATCase catalyzes the formation of N-carbamoylaspar-tate from aspartate and carbamoyl phosphate, and is the committed step in pyrimidine biosynthesis (Fig. 2). The binding of the two substrates aspartate and carbamoyl phosphate is cooperative, as shown by the sigmoidal curve of V0 against substrate concentration (Fig. 3). 

Unlike in purine biosynthesis, the pyrimidine ring is synthesized before it is conjugated to PRPP. The first reaction is the conjugation of carbamoyl phosphate and aspartate to make N-carbamoylaspartate. The carbamoyl phosphate synthetase used in pyrimidine biosynthesis is located in the cytoplasm, in contrast to the carbamoyl phosphate used in urea synthesis, which is made in the mitochondrion. The enzyme that carries out the reaction is aspartate transcarbamoylase, an enzyme that is closely regulated. 

ATCase and ornithine carbamoyltransferase (OTCase) catalyze analogous reactions. ATCase transfers the carbamoyl moiety from carbamoyl phosphate to aspartate, and OTCase transfers the carbamoyl moiety from carbamoyl phosphate to ornithine. They both share a common N-terminal functional domain, which binds carbamoyl phosphate. The C-terminal domains of these enzymes are structurally similar but have... 

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.

Figure 25.2. de Novo Pathway for Pyrimidine Nucleotide Synthesis. The C-2 and N-3 atoms in the pyrimidine ring come from carbamoyl phosphate, whereas the other atoms of the ring come from aspartate. 

Figure 10.1 ATCase reaction. Aspartate transcarbamoylase catalyzes the committed step, the condensation of aspartate and carbamoyl phosphate to form N Carbamoylaspartate, in pyrimidine synthesis.

Stevens, R. C, Gouaux, J. E., and Lipscomb, W. N. 1440. Structural consequences of effector binding to llic state of aspartate carbamoyl transfer a sc Crystal structures ol the unligaterl and. A4 P-... 

Aspartate transcarhamoylase. Write the mechanism (in detail) for the conversion of aspartate and carbamoyl phosphate into N-carbamoyiaspartate. Include a role for the histidine residue present in the active site. 

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. 

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