Orotidylic acids biosynthesis

A common intermediate for all the nucleotides is 5-phosphoribosyl-l-diphosphate (PRPP), produced by successive ATP-dependent phosphorylations of ribose. This has an a-diphosphate leaving group that can be displaced in Sn2 reactions. Similar Sn2 reactions have been seen in glycoside synthesis (see Section 12.4) and biosynthesis (see Box 12.4), and for the synthesis of aminosugars (see Section 12.9). For pyrimidine nucleotide biosynthesis, the nucleophile is the 1-nitrogen of uracil-6-carboxylic acid, usually called orotic acid. The product is the nucleotide orotidylic acid, which is subsequently decarboxylated to the now recognizable uridylic acid (UMP). 

In the next step of pyrimidine biosynthesis, the entire aspartate molecule adds to carbamoyl phosphate in a reaction catalyzed by aspartate transcarbamoylase. The molecule subsequently closes to produce a ring (catalyzed by dihydroorotase), which is oxidized to form orotic acid (or its anion, orotate) through the actions of dihydroorotate dehydrogenase. The enzyme orotate phosphoribosyl transferase catalyzes the transfer of ribose 5-phosphate from PRPP to orotate, producing orotidine 5 -phosphate, which is decarboxylated by orotidylic acid dehydrogenase to form... 

Five of the enzymes of UMP biosynthesis exist in the soluble fraction of Ehrlich ascites carcinoma as two enzyme complexes [143]. One complex contains the first three enzymes of the pathway, carbamoyl phosphate synthetase, aspartate carbamoyltransferase and dihydro-orotase and has an apparent molecular weight of 800000 to 850000 daltons. The second enzyme complex contains orotate phosphoribosyltransferase and orotidylic acid decarboxylase and sediments in a sucrose gradient with 30% dimethyl sulphoxide and 5% glycerol with an apparent molecular weight of 105 000 to 115000 daltons [143]. 

A metabolic alteration leading to overproduction of either PRPP or glutamine would cause an overproduction of 5 -phosphoribosylamine. Excess PRPP could result from overproduction of its precursor glucose, or from reduced use in other pathways, such as pvrimidine biosynthesis. Increased incorporation of " Qglucose and accelerated turnover of PRPP have been reported. A case of gout with low orotidylic acid pyrophosphorylase and decarboxylase activity has also been described. Whether these metabolic changes constitute the primary injury or are several steps removed from it remains to be established. 

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 riboside of orotic acid, orotidine, was isolated subsequently from the culture medium of a uridine-requiring Neurospora mutant (350). Orotidine was readily split to orotic acid because of unusually great acid lability this offered a plausible explanation for previous failures to isolate a conjugated form of orotic acid from those natural sources that 3oelded the free acid (338, 342, 343). With the current knowledge of the individual steps in pyrimidine biosynthesis (Section VI, D, 2), it is likely that orotidine was derived from orotidylic acid (orotidine 5 -phosphate), and that a genetic block in the mutant organism prevented decarboxylation of oro-... 

Even though orotidylic acid or orotidine was implicated in pyrimidine formation, the precise role of orotic acid per se remained to be evaluated. On the other hand, it was posable that orotic acid was a normal intermediate that condensed with a ribose compound to yield orotidine or orotidylic acid during the biosynthetic process. In support of this thesis, it was found that 5-phosphoribosyl-l-pyrophosphate was utilized for nucleotide formation from orotic acid (83). On the other hand, it was equally posable that an aliphatic compound, such as aminofumaric acid (335) or carbamylaspartic acid (339), could have coupled with a ribose compound and formed orotidine or orotidylic acid directly without the existence or participation of orotic acid per se. In this latter instance, orotic acid would not be conadered a true intermediate in pyrimidine biosynthesis but merely an accidental cleavage product of hi ly labile orotidine or orotidylic acid. At this time research in the area of purine biosynthesis indicated that a series of acyclic intermediates attached to ribose 6-phosphate were biosynthetic intermediates and that free purines per se were not (Section II, B.). 

The details of the biosynthesis of purines and pyrimidines are discussed in Chapter 18. An interesting point of difference in the synthesis of the purine and pyrimidine ring systems is that the purine ring is synthesized as part of a nucleotide (8) whereas the parent pyrimidine, orotic acid, is synthesized as such. The element of pentose-phosphate is added to the completed pyrimidine ring to form the nucleotide orotidylic acid. Oro-tidylic acid is then decarboxylated to yield uridylic acid (da). 

Many potent pyrimidine antagonists have also been obtained by isosteric replacement in the heterocyclic ring. Oxonic acid (Fig. 12) an analogue of orotic acid, prevents the conversion of the latter to orotidylic acid (Fig. 2). A later stage of pyrimidine biosynthesis, the decarboxylation of orotidylic acid to form uridylic acid (Fig. 2) is strongly inhibited by 6-azauracil (Fig. 12 this chemical should be correctly termed 4-azauracil). The riboside of 6-azainacil acts on the same pathways as the base, but its inhibition of the orotidylate carboxylase enzyme is some 20 times more potent... 

The first step in de novo pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS), and the bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP (adenine dinucleotide phosphate). Ammonia then reacts with carboxyphosphate to form carbamic acid. The latter is phosphorylated by another molecule of ATP with the mediation of CPS to form carbamoyl phosphate, which reacts with aspartate by aspartate transcarbamoy-lase to form A-carbamoylaspartate. The latter cyclizes to form dihydroorotate, which is then oxidized by NAD-1- to generate orotate. Reaction of orotate with 5-phosphoribosyl-l-pyrophosphate (PRPP), catalyzed by pyrimidine PT, forms the pyrimidine nucleotide orotidylate. This reaction is driven by the hydrolysis of pyrophosphate. Decarboxylatin of orotidylate, catalyzed by orotidylate decarboxylase, forms uridylate (uridine-5 -monophosphate, UMP), a major pyrimidine nucleotide that is a precursor of RNA (Figure 6.53). 

The final steps of pyrimidine biosynthesis novo which are catalyzed by two sequential enzymes, orotate phosphoribosyltransfer-ase (OPRT) and orotidylic decarboxylase (ODC), involve the PP-ribose P dependent conversion of orotic acid to orotidine-5 -monophosphate (OMP) followed by decarboxylation at the 7 position to form uridine 5 -monophosphate (UMP) (Fig. 1). UMP is then utilized further in the synthesis of nucleic acids and co-enzymes. Defects at this site in this metabolic pathway are important for they can result in "pyrimidine starvation" from depletion of the intracellular pool of pyrimidine nucleotides. In man the rare genetic disease, orotic aciduria, involves a deficiency of both OPRT and ODC (Type 1) (Smith, Sullivan and Huguley, 1961) or, less commonly, only ODC (Type II) (Fox, 0 Sullivan and Firken, 1969). 

In reversal experiments using the human B-cell line (RPMI 8422), pyrazofurin inhibited cell growth at a concentration of 10 M. This PF effect was completely reversed with the addition of 10 M to 10 M of uridine or cytidine but not by orotic acid or orotidine. This is additional confirmation of the assumption that the site of inhibition in the biosynthesis of pyrimidine nucleotides is at the site of orotidylic carboxylase ... 

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... 

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