Orotidylic decarboxylase

The orotic aciduria that accompanies Reye s syndrome probably is a consequence of the inabifity of severely damaged mitochondria to utifize carbamoyl phosphate, which then becomes available for cytosofic overproduction of orotic acid. Type I orotic aciduria reflects a deficiency of both orotate phosphoribosyltransferase and orotidylate decarboxylase (reactions 5 and 6, Figure 34—7) the rarer type II orotic aciduria is due to a deficiency only of orotidylate decarboxylase (reaction 6, Figure 34-7). 

OROTIDYLATE DECARBOXYLASE Orotidine-5 -phosphate decarboxylase, OROTIDYLATE DECARBOXYLASE Orotidine-5 -phosphate pyrophosphorylase,... 

These experiments suggest that, in the presence of 6-azauracil, there is a block in the utilization of pyrimidines at the nucleotide level. Later experiments have shown that there is an inhibition of orotidylic decarboxylase... 

At this stage, orotate couples to ribose, in the form of 5-phosphoribosyl-l-pyrophosphate (PRPP), a form of ribose activated to accept nucleotide bases. PRPP is synthesized from ribose-5-phosphate, formed by the pentose phosphate pathway, by the addition of pyrophosphate from ATP. Orotate reacts with PRPP to form orotidylate, a pyrimidine nucleotide. This reaction is driven by the hydrolysis of pyrophosphate. The enzyme that catalyzes this addition, pyrimidine phosphoribosyltransferase, is homologous to a number of other phosphoribosyltransferases that add different groups to PRPP to form the other nucleotides. Orotidylate is then decarboxylated to form uridylate (IMP), a major pyrimidine nucleotide that is a precursor to RNA. This reaction is catalyzed by orotidylate decarboxylase. 

Orotidylate decarboxylase is one of the most proficient enzymes known. In its absence, decarboxylation is extremely slow and is estimated to take place once every 78 million years with the enzyme present, it takes place... 

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. 

Orotate condenses with PRPP in a reaction catalyzed by orotate phosphoribosyl transferase to form the nucleotide orotidylate (OMP). Orotidylate decarboxylase converts OMP to the more abundant nucleotide UMP. The reaction occurs during de novo pyrimidine biosynthesis and is therefore not a salvage reaction. 

Estimates of PRibPP levels from studies with fresh cell lysates indicate that the concentration is about 8 X 10 M which is well below the Km of the enzyme 2 X 10 M (D3, H5). The assay techniques are indirect. In one, C-labeled adenine is added along with excess purified APRT. The amount of AMP- C formed is a measure of the PRibPP present. In a second assay, carboxy-labeled orotic acid is added together with orotidylate pyrophosphorylase and orotidylate decarboxylase. The liberated C02 is a measure of the orotidylic acid which is a measure, in turn, of the PRibPP concentration. 

Azauracil (4.41) is used in agriculture as a fungicide to inhibit powdery mildew (e.g. in cucumbers). It is converted in the fungal cell to the ribotide which, being an analogue of orotidylic acid, blocks orotidylic decarboxylase (Dekker, 1968). Although a side effect on the central nervous system precluded its use in Man, the triacetyl-derivative of this ribotide, azaribine (see Section 3.6, p. 102), has proved valuable in the oral treatment of psoriasis (Calabresi and Turner, 1966). 

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). 

C6 Campbell, M. T., Gallagher, N. D. and O Sullivan, W. J. Multiple molecular forms of orotidylate decarboxylase from human liver. Biochem. Med., 17, 128-140 (1977)... 

The irreversible decarboxylation step, catalyzed by orotidylate decarboxylase, has been demonstrated in various animal tissues. 

Bresnick (52) has concluded that in the partially hepatectomized rat, during the first 12 hours of liver regeneration, the activity of orotate phosphoribosyltransferase determines the rate of synthesis of the uridine phosphates. The potential activity of orotidylate decarboxylase is in excess of that of orotate phosphoribosyltransferase. This probably accounts for the virtual absence of orotidylate in animal tissues. The decarboxylation step is irreversible and may be subject to feedback inhibition by uridylate, which is a competitive inhibitor of the enzyme in rat liver and yeast (19). 

In cells, azauridine is converted to the 5 -monophosphate, which is a competitive inhibitor of orotidylate decarboxylase. Orotidylate accumulates because of the inhibited enzyme and its subsequent degradation explains the urinary excretion of orotidine in azauridine-treated animals. Azauridine has been used to treat acute leukemia in man, but resistance to the drug develops rapidly. 

It is noteworthy that both cytidine and uridine are substrates for this enzyme, whereas the corresponding deoxyribosides are not. There does not appear to be a separate cytidine kinase. It will be noted that orotidine is not a substrate for uridine kinase. Although orotidine is not ordinarily present in appreciable amounts in tissues, it appears, along with orotate, in the urine of animals treated with azauridine (see Chapter 11), and orotidine accumulates in the culture medium of certain Neurospora mutants which lack orotidylate decarboxylase. Orotidine is most likely a breakdown product of orotidylate and the provision in nature of an appropriate kinase activity would appear unnecessary. 

Weissmann et al. (4) have calculated that in man approximately 800 to 1000 mg of uracil are synthesized de novo per day. The normal urinary excretion of orotate is approximately 1.4 mg per day, and that of orotidine, 2.5 mg per day (6). In patients with orotic aciduria due to decreased orotidylate decarboxylase, the excretion of orotate may increase 20-fold (5), although the excretion of orotidine does not necessarily increase. 

CioHi3N20,iP 368.193 Formed in the biosynthetic pathway in yeast. Decarboxylation by Orotidylate decarboxylase affords Uridine 5 -phos-phate which is the route to Uridine and its derivatives de novo and consequently one of the most important processes in nucleic acid synthesis. Trihydrate (as Na salt). Moffatt, J.G. et al., J.A.C.S., 1963, 85, 1118 (synth)... 

This is in agreement with our results obtained in human cultured lymphoblasts. Oxipurinol inhibits orotidyl decarboxylase(ODC), Purines might inhibit the conversion of orotic acid to orotidine monophosphate by lowering the intracellular PRPP-concentration (Crandall et al. 1978). 

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). 

Fig. 4. Metabolism of allopurinol and oxipurinol to ribonucleotides that act as inhibitors of orotidylic decarboxylase. OPRT = orotate phosphoribosyltransferase HGPRT = hypoxanthine-guanine phospho-ribosyltransferase.

Fig. 5. The effect of allopurinol therapy on erythrocyte orotidylic decarboxylase (ODC), orotate phosphoribosyltransferase (OPRT), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and adenine phosphoribosyltransferase (APRT), and serum urate in three patients with gout. The upper limits of normal OPRT and ODC activity in erythrocytes (mean 2 S.D.) are indicated by the dotted and solid horizontal lines, respectively. (From Beardmore, Cashman and Kelley, 1972).

Fig. 6. Orotate phosphoribosyltransferase (OPRT) and orotidylic decarboxylase (ODC) in circulating erythrocytes of different density after initiation of therapy with allopurinol (800 mg/day) in patient C.R. Mean activity is plotted against specific gravity (increasing values correspond to increasing cell age in vivo). Control, allopurinol therapy (solid lines) day 6, day 9 and 13, - ...

In an accompanying paper Dr. Elion will present evidence for the formation in vivo of alio- and oxipurinol ribonucleotides, their characterization and their quantification in tissues after various dosage regimens. It is the purpose of this paper to report the kinetics of their inhibitory activities on a representative orotidylate decarboxylase vitro, and the effects on pyrimidine metabolism consequent to their formation vivo. 

Orotidylate decarboxylase is a key enzyme in the pathway of pyrimidine nucleotide biosynthesis de novo (Fig. 2) ... 

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