Carbamoyl aspartate

One of the steps in the biosynthesis of uridine monophosphate is the reaction of aspartate with carbamoyl phosphate to give carbamoyl aspartate followed by cyclization to form dihydroorotate. Propose mechanisms for both steps. 

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).

A different, simpler , pathway is involved in the synthesis of pyrimidine nucleotides. A pyrimidine base (orotate), is synthesised first. Then the ribose is added from 5-phosphoribosyl 1-pyrophosphate. The two precursors for the formation of orotate are carbamoylphosphate and aspartate, which form carbamoyl aspartate, catalysed by aspartate carbamoyltransferase. 

Purine Biosynthesis Is Regulated at Two Levels Pyrimidine Biosynthesis Is Regulated at the Level of Carbamoyl Aspartate Formation Deoxyribonucleotide Synthesis Is Regulated by Both Activators and Inhibitors... 

Pyrimidine Biosynthesis Is Regulated at the Level of Carbamoyl Aspartate Formation... 

In bacteria, the first committed step in pyrimidine nucleotide biosynthesis is the formation of carbamoyl aspartate... 

The methylene group prevents the elimination of a phosphate, which is required to convert the postulated intermediate into carbamoyl aspartate. The suggested oxygen analog might eliminate phosphate (i.e., be a substrate for aspartate car-bamoyltransferase). 

Aspartate carbamoyltransferase catalyzes the formation of carbamoyl aspartate from carbamoyl phosphate and aspartate in the first committed step of pyrimidine biosynthesis (Chap. 15). The enzyme from the bacterium E. coli (Mr = 310,000) consists of 12 subunits, six regulatory and six catalytic. CTP is a negative effector i.e., it inhibits the enzyme, and does so through binding to the regulatory subunits. ATP is a positive effector that acts through the regulatory subunits, while succinate inhibits the reaction by direct competition with aspartate at the active site (see Chap. 9 for more on effectors). 

For dihydroorotate synthetase, the product of reaction 1, carbamoyl phosphate (CAP) is very unstable but is rapidly transformed by aspartate transcarbamoylase which is 50 times more active (per active site) than carbamoyl phosphate synthetase. High levels of carbamoyl aspartate (CA-asp) may be toxic, but this intermediate is rapidly consumed by the high dihydroorotase activity. Because the first three reactions are catalyzed by a single protein, the three enzyme active sites are expressed in a constant ratio under all conditions of growth this maintains CAP and CA-asp at low levels. For UMP synthase, OMP decarboxylase is far more active (per active site) than orotate PRTase, resulting in low cellular levels of the intermediate, OMP, which would otherwise be subject to enzymatic hydrolysis (in cells from higher animals). 

Note CAA = Carbamoyl Aspartate DHOA = Dihydroorotate OMP = Orotidine Monophosphate... 

In the biosynthesis of both pyrimidine and urea (or arginine) (Chapter 17), carbamoyl phosphate is the source of carbon and nitrogen atoms. In pyrimidine biosynthesis, carbamoyl phosphate serves as donor of the carbamoyl group to aspartate with the formation of carbamoyl aspartate. In urea synthesis, the carbamoyl moiety of carbamoyl phosphate is transferred to ornithine, giving rise to citrulline. 

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. 

Zn > Co > Cd. Additionally, DHO activity is dependent on a single ionization for the dihydroorotate (favorable deprotonation) and carbamoyl aspartate (favorable protonation) substrates with apparent pA), values of 6 and 8.2, respectively. The pi), values change upon substitution with Co, suggesting that this reflects a metal-dependent ionization. There is a significant deuterium solvent isotope effect observed in /feat with the dihydroorotate ( cat / cat = 2.5 (ifcat/ M) /( cat/ M) = 1-1) and thio-dihydroorotate = 2.3 ... 

Aspartate + Carbamoyl Phosphate <=> Carbamoyl aspartate (catalyzed by Aspartate T ranscarbamoylase)... 

Carbamoyl aspartate is a metabolite in the pathway of pyrimidine biosynthesis. 

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