Pyrimidine 5 -orotidylic acid

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). 

By contrast, if a pyrimidine is to be made, Nature assembles a general pyrimidine structure first and adds it in one step to the PRPP molecule, again in an S>j2 reaction using a nitrogen nucleophile. This general nucleotide, orotidylic acid, can be converted into the other pyrimidine nucleotides by simple chemistrv. 

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... 

Orotidyhc acid decarboxylase (orotidine 5 -phosphate carboxy-lyase, EC 4.1.1.23) catalyses the only irreversible step in the pyrimidine synthesis de novo. The enzyme is competitively inhibited by UMP and CMP [114-116] and some anomalous pyrimidine nucleoside 5 -monophosphates. The activity of orotidylic acid decarboxylase in excess of that of orotate phosphoribosyltransferase accounts for the absence of orotidine 5 -phosphate in the pool of low molecular weight compounds in animal cells. 

Although allopurinol and oxipurinol are potent inhibitors of UMP synthesis [120,131] through the inhibition of orotidylic acid decarboxylase (oxipurinol with a 2,4-diketo pyrimidine ring is capable of acting as an analogue of orotic acid, and 1-ribosyl-oxipurinol 5 -phosphate [132] is a... 

There are several synthetic derivatives of orotic acid and pyrimidine analogues which, after their conversion, interfere with the activity of orotidylic acid decarboxylase [263,264]. i ile 6-azacytidine 5 -phosphate is only one tenth as active as 6-azauridine 5 -phosphate [265], 5-hydroxyuridine 5 -phosphate [266] and aminouridine 5 -phosphate [267] are potent inhibitors of orotidylic acid decarboxylase. The inhibitory action of allopurinol and of its metabolites on pyrimidine synthesis de novo [268] was mentioned in Chapter 3. 

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. 

It is well established that aza analogs of purines, pyrimidines and their nucleosides possess significant but varying potency as antineoplastic agents [1-7]. Thus, for example, 6-azacytidine is an inhibitor for orotidylic acid decarboxylase [3] and 6-azauracil inhibits the development of animal tumors [4] and human acute leukemia [5] similarly, 8-azaguanine is a highly effective anti-neoplastic agent [6] and also inhibits animal tumors [7]. 

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 formation of the first nucleotide in the pyrimidine sequence, orotidylic acid (orotidine 5 -phosphate), was accomplished by the reaction of 5-phosphoribosyl-l-pyrophosphate (PRPP) with orotic acid 83) (Fig. 22). Other pyrimidines and carbamylaspartic acid did not react with PRPP in the presence of the enzyme, which has been named orotidine 5 -phosphate pyrophosphorylase. Several purine analogs, e.g., 6-uracilsulfonic acid, 6-uracil methyl sulfone, which inhibited the growth of several organisms (S78, 379), probably inhibited the formation of orotidylic acid. 

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). 

Fluorine has the closest atomic dimensions to the hydrogen atom, and pyrimidine analogues where hydrogen is substituted by fluorine are powerful antimetabolites. 5-fluoro-orotic acid (Fig. 12), for instance, prevents the conversion of orotic acid to orotidylic acid (Fig. 2) and the methylation of deoxyuridylic acid to form thymi-dylic acid (Fig. 9). 

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 answer is e. (Murray, pp 375-401. Scriver, pp 2663-2704. Sack, pp 121-138. Wilson, pp 287—320.) Orotic aciduria is the buildup of orotic acid due to a deficiency in one or both of the enzymes that convert it to UMP Either orotate phosphoribosyltransferase and orotidylate decarboxylase are both defective, or the decarboxylase alone is defective. UMP is the precursor of UTP, CTP, and TMP All of these end products normally act in some way to feedback-inhibit the initial reactions of pyrimidine synthesis. Specifically, the lack of CTP inhibition allows aspartate transcarbamoylase to remain highly active and ultimately results in a buildup of orotic acid and the resultant orotic aciduria. The lack of CTP, TMP, and UTP leads to a decreased erythrocyte formation and megaloblastic anemia. Uridine treatment is effective because uridine can easily be converted to UMP by omnipresent tissue kinases, thus allowing UTP, CTP, and TMP to be synthesized and feedback-inhibit further orotic acid production. 

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). 

With in vivo experiments, Hurlbert and Potter ) first showed that in rat liver, uridine nucleotides were intermediates in the conversion of orotate to nucleic acid pyrimidines the first of the three uridine phosphates to become labeled in this process was the monophosphate, uridylate (UMP) IS). The synthesis of uridylate from orotate takes place in two steps (a) the condensation of orotate with PP-ribose-P to form orotidylate (orotidine 5 -monophosphate, or OMP), and (b) decarboxylation of orotidylate. 

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... 

---