Pyrimidine Carbamyl phosphate

Urinary orotic acid generally is very elevated in babies with OTC deficiency and normal or even low in the infant with CPS deficiency. Patients with OTC deficiency have orotic aciduria because carbamyl phosphate spills into the cytoplasm, where it enters the pathway of pyrimidine synthesis. 

A second, cytosolic CPS activity (CPSII) occurs in mammals as part of the CAD trifunctional protein that catalyzes the first three steps of pyrimidine synthesis (CPSII, asparate tran-scarbamoylase, and dihydroorotase). The activities of these three enzymes—CPSII, aspartate transcarbamoylase, and dihydroorotase—result in the production of orotic acid from ammonium, bicarbonate, and ATP. CPSII has no role in ureagenesis, but orotic aciduria results from hepatocellular accumulation of carbamyl phosphate and helps distinguish CPSI deficiency from other UCDs. Defects in CPSI classically present with neonatal acute hyperammonemic encephalopathy. The plasma citrulline and urine orotic acid concentrations are both low. A definitive diagnosis can be established by enzyme assay of biopsied liver tissue or by mutation analysis. 

Figure 9-1 Sites of feedback inhibition in carbamyl phosphate metabolism of E. coli. Note that aspartate trascarbamylase is the first enzyme on the unique pathway to pyrimidine compounds.

The role of ATP in the carboxylation of biotin is unclear. It is possible that biotin is O-phosphorylated during the carboxylation reaction. However, evidence suggests that the immediate reactive species that carboxylates biotin is carboxyphosphate, as in the (biotin-independent) reaction of carbamyl phosphate synthetase in urea and pyrimidine synthesis. 

Methylating agents can be generated by chemical ni-trosation of endogenous metabolites. For example, methylamine produced by the decomposition of organic matter can condense with carbamyl phosphate, a precursor of pyrimidines, to form methylurea, which in turn can be nitrosated to yield methylnitrosourea (MNU). Such nitrosation reactions can be catalyzed by bacterial enzymes (35). 

The condensation of carbamyl phosphate and L-aspartate, catalyzed by aspartate trans-carbamoylase (ATCase), produces iV-carba-myl-L-aspartate (Equation 17.38). This is one of the early steps in de novo pyrimidine biosynthesis, also a requirement for cell division. 

FIGURE 9.7 Pyrimidine biosynthetic pathway. The pathway of pyrimidine biosynthesis involves six steps and results in the production of uridine 58-monophosphate. Folate is not used in this pathway. The pathway commences with the transfer of the amide nitrogen of glutamine to bicarbonate to produce carbamyl phosphate. This molecule then reacts with aspartate to form the beginnings of the six-membered pyrimidine ring. 

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). 

Hi. Hager, E. E., and Jones, M. E., A glutamine-dependent enzyme for the synthesis of carbamyl phosphate for pyrimidine biossmthesis in fetal rat liver. J. Biol. Chetn. 242, 5674-5680 (1967). 

Tl. Tatibana, M., and Ito, K., Carbamyl phosphate synthetase of the hematopoietic mouse spleen and the control of pyrimidine biosynthesis. Biochem. Biophys. Res. Commun. 26, 221-227 (1967). 

W5. Williams, L. G., and Davis, R. H., Pyrimidine-specific carbamyl phosphate synthetase in neurospora crassa. J. Bacteriol. 103, 335-341 (1970). 

The synthesis of nucleotide triphosphates required for polynucleotide chain building is a complex process which will not be considered in full detail here. The biosynthetic routes for purine and pyrimidine nucleosides are somewhat different and commence with 5 phosphoribosyl-l-pyrophos-phate and carbamyl phosphate, respectively. These two materials undergo successive enzyme-catalysed reactions, linking at times with compounds encountered in other biochemical cycles, and utilising ATP in several stages. Polynucleotides can be synthesised by purely chemical means in the laboratory (Chapter 10.4). 

Pyrimidine biosynthesis commences with a reaction between carbamyl phosphate and aspartic acid to give carbamyl aspartic acid which then nndergoes ring closure and oxidation to orotic acid. A reaction then occurs between orotic acid and 5-phosphoribosyl pyrophosphate to give orotidine-5-phosphate which on decarboxylation yields uridine-5-phosphate (UMP). By means of two successive reactions with ATP, UMP can then be converted into UTP and this by reaction with ammonia can give rise to cytidine triphosphate, CTP (11.126). 

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-... 

In addition to the requirement for pyrimidine nucleotide synthesis, carbamyl phosphate is required for synthesis of arginine and urea. Carbamyl phosphate synthesis is a prominent activity in ureotelic liver and is aimed primarily at the formation of urea the process of urea synthesis is served by a special carbamyl phosphate synthetase which is quite distinct from the enzymes responsible for carbamyl phosphate synthesis in extrahepatic tissues and in the livers of uricotelic animals. A third mechanism for synthesis of carbamyl phosphate is found in bacteria. 

Pi rard et al. (43) have demonstrated that the synthesis of carbamyl phosphate synthetase II in E. coli is under repressor control and that a single species of this enzyme serves both the arginine and pyrimidine pathways. Uridylate is a negative effector of the synthetase and ornithine reverses this inhibition (44)- The existence of two separate Type II synthetases has been demonstrated in yeast, one each serving the arginine and pyrimidine pathways (45). 

Figure 13-1 shows that reduction of the 5 6 double bond is the first step in the breakdown of the pyrimidine ring. This is followed by oxidative cleavage of the 3 4 bond to form ureidopropionic or ureidoisobutyric acid, from which /3-amino acids are formed with the release of NHj and CO. Whether carbamate or carbamyl phosphate is the primary product is not known. The /3-amino acids are then oxidatively deaminated to /3 dehydes, which are converted to acids which are variously metabolized. 

As early as 1949, it was demonstrated that injected or " C-labeled orotic acid was readily incorporated into DNA and RNA of mammalian tissue, indicating that orotic acid is a precursor of nucleic acid pyrimidine. The next step in pyrimidine biosynthesis is the formation of the first nucleotide in the sequence. It involves the reaction between ribosyl pyrophosphate and orotic acid to yield 5 -orotidylic acid the reaction is catalyzed by orotidylic pyrophosphorylase. Thus, the first steps of pyrimidine biosynthesis differ from the early steps of purine biosynthesis in at least two ways. Orotic acid, instead of being synthesized atom by atom as is the case for the purine ring, is made from the condensation of rather large molecules, namely, carbamyl phosphate and aspartic acid. Furthermore, all the steps of purine biosynthesis occur at the level of the nucleotide, but the the pyrimidine ring is closed at the level of the base. 

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 cycle starts with carbamyl phosphate formation (this reaction was discussed in the section on pyrimidine biosynthesis). Carbamyl phosphate synthetase catalyzes the condensation of active CO2 with NH4 to yield carbamyl phosphate, a precursor of pyrimidines and urea. 

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. 

The carbon in the l-position and the nitrogen in the 2-position of the pyrimidine ring are derived from NHj and CO a which react in the presence of ATP to form carbamyl phosphate. 

The carbamyl phosphate is condensed with a molecule of aspartate giving ureidosuccinic acid, from which orotic acid is formed by cyclization and oxidation. In the presence of PRPP and a pyrophosphorylase this acid forms a ribotide and decarboxylation yields uridine monophosphate. The decarboxylation of the product of amination of orotidine phosphate gives cytidine monophosphate (Fig. 75). It can be seen that the pentose intermediate in pyrimidine nucleotide biosynthesis is PRPP, the same as for purine nucleotide biosynthesis. 

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. 

In preceding sections of this chapter, the important metabolic reactions which yield ammonia have been discussed. Certain of these systems are capable of fixing ammonia (glutamic dehydrogenase, alanine dehydrogenase, L-amino acid oxidase, etc.). The fixation of ammonia in the glutamine synthetase system will be discussed in Chapter 17. The present section will deal with (a) enzymes which fix ammonia to form carbamyl phosphate and (b) enzymes which utilize carbamyl phosphate for the synthesis of arginine (and urea) and pyrimidines. 

It should be noted that the synthesis of carbamyl phosphate provides an important precursor for two metabolic pathways, one leading to the synthesis of the amino acid arginine and the other leading to the synthesis of pyrimidines via orotic acid. 

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