Carbamyl aspartic acid, biosynthesis

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). 

The operation of the feedback mechanism in the cell can be described in the following manner if nucleic acid syntheris were proceeding at a rapid rate, the concentration of nucleotides in the cell, e.g. CMP, would decrease, relieve aspartate-carbamyl transferase (also called carbamyl-aspartic acid synthetase) reaction of any inhibition, and thus permit an increased rate of pyrimidine biosyntheris. If nucleic acid syntheris were slowed or halted, the CMP concentration would rise, inhibit the enzyme, and thereby decelerate pyrimidine biosynthesis. 

Figure 2 shows the main stages of pyrimidine nucleotides biosynthesis. In stage 1 aspartic add and carbamyl phosplmte (formed from ammonia, CO3 and ATP) condense to form carbamyl aspartic acid. This derivative cydizes with loss of water to form dihydroorotic add which is converted by a dehydrogenase to orotic add. The nudeotide of orotic acid (orotidylic add (OI )) is dien formed in... 

Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is an allosteric enzyme which controls the first step of p5oimidine de novo biosynthesis. It catalyzes the condensation of L-aspartic acid with carbamyl phosphate to produce carbamoyl-L-aspartate... 

Historically, carbamyl aspartate was recognized as a likely intermediate in pyrimidine biosynthesis because (a) this compound is an assembly of two elementary precursors of the pyrimidine ring, (b) carbamyl aspartate would satisfy the nutritional requirement of L. bvlgaricus 09 for orotate, and (c) labeled carbamyl aspartate was incorporated into ribonucleic acid pyrimidines in L. bulgaricus and, as well, served as an orotate precursor in liver slice trapping experiments such as those mentioned above. 

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. 

An early form of therapy involves eliminating the substrate either by excluding the substrate from the diet, as in phenylketonuria, or by administering drugs—such as penicillamine in Wilson s disease or allopurinol in gout. Orotic aciduria can be corrected by the administration of uridine, which provides the substrate for the biosynthesis of the nucleosides used in RNA and DNA synthesis and is also a substrate for the biosynthesis of inhibitors of the carbamyl aspartate synthetase, the first enzyme in the formation of orotic acid. By this feedback inhibition, the levels of orotic acid in the urine are reduced by the administration of uridine. 

The Biosynthesis of the Pyrimidine Ring begins with aspartic acid and carbamyl phosphate. The latter is an energy-rich compound which reacts with the former to give carbamylaspartic acid. Ring closure consumes ATP and is in principle an acid amide formation (peptide synthesis). The intermediate dihydro-orotic acid is dehydrogenated to orotic acid, probably by action of a flavoprotein. Orotic acid is the key precursor of pyrimidine nucleotides. It reacts with phosphoribosyl pyrophosphate. The removal of pyrophosphate yields the nucleotide of orotic acid, whose enzymic decarboxylation produces uridine 5 -phosphate. Phosphorylation with ATP yields uridine pyrophosphate and, finally, uridine triphosphate. Beside the above pathway, there is the further possibility of converting free uracil and ribose 1-phosphate to the nucleoside and from there with ATP to the nucleotide. 

In other cases we encounter not induction but repression of enzyme synthesis by a metabolite in whose synthesis the enzyme is concerned (the same may also be inhibited in its activity by other components of the reaction sequence in which it it involved, see p. 252). The enzyme whose activity is lost or markedly reduced is not necessarily that which completes the synthesis of the repressor molecule (i.e. not that catalysing the last reaction of the biosynthetic pathway). For instance, uracil can suppress, in certain strains of the bacterium, Escherichia coU, the activity of the enzyme aspartate carbamyl-transferase which promotes the interaction between aspartic acid and carbamyl phosphate, a reaction which is the first step in the reaction sequence involved in pyrimidine biosynthesis. Further, the experimental evidence indicates that the uracil acts as a repressor by preventing synthesis of the enzyme. 

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. 

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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