Orotidylate decarboxylase activity

Orotidylate decarboxylase activity in leukocytes. Since mass spectrometry played no role in this phase of our work, results will only be summarized here for completeness. PF resulted in more than 90% inhibition of the enzyme activity in white blood cells in the first few days within a week to 10 days activity returns to normal. Both bolus and infusion administration resulted in the same type and extent of inhibition. 

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. 

Figure 27-27), aspartate transcarbamoylase, and dihydroorotase activity. Each subunit of Pyr 1-3 has a molecular weight of 200,000-220,000, and the native enzyme exists as multiples of three subunits. The second gene codes for dihydroorotate dehydrogenase which is located on the outer side of the inner mitochondrial membrane. Dihydroorotate, the product of Pyr 1-3, passes freely through the outer mitochondrial membrane and converted to orotate. Orotate readily diffuses to the cytosol for conversion to UMP. The third gene codes for another multifunctional polypeptide known as UMP synthase (Pyr 5,6). Pyr 5,6 (M.W. 55,000) contains orotate phosphoribosyltransferase and orotidylate (orotidine-5 -monophosphate) decarboxylase activity. Use of multifunctional polypeptides is very efficient, since the intermediates neither accumulate nor become consumed in side reactions. They are... 

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. 

H34 Hoffman, D. H. and Sweeney, M. J. Orotate phosphor-ibosyl transferase and orotidylic acid decarboxylase activities in liver and Morris hepatomas. Cancer Res., 33, 1109-1112 (1973)... 

By fractionating yeast extracts, preparations of the orotate phos-phoribosyltransferase were obtained which were free of the decarboxylase activity 14). When incubated with PP-ribose-P and orotate, these preparations formed orotidylate this product was identical with orotidylate prepared by the enzymatic phosphorylation of orotidine. The stoichiometry of the reaction was also established. 

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). 

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. 

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. 

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. 

The cells were incubated with 8 x 10 M of pyrazofurin for one hour at 37 C. This concentration is about 10 times that needed for the inhibition of 90% of the activity of orotidylate decarboxylase in L1210 cells (15). After incubation, cells were washed twice with 0.15 M NaCl to remove free pyrazofurin. Next 5 volumes of 1 N perchloric acid was added to the cell pellets and centrifuged for 15 min to remove the precipitate. The acid-soluble portion of the extract was neutralized with concentrated KOH and centrifuged to remove the precipitated potassium perchloride. The resulting extract was evaporated to total dryness under nitrogen stream (dry, room temperature), and the resulting residue (somewhat brownish in appearance, probably due to some charring) was derivatized as described later. 

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. 

There are several studies on the effect of allopurinol and its metabolic derivatives on orotate phosphoribosyltransferase and orotidylic acid decarboxylase [127-129]. The administration of allopurinol to rats results in an increased urinary excretion of orotic acid and orotidine [127,130,131], and in elevated activities of orotate phosphoribosyltransferase and orotidylic acid decarboxylase in erythrocytes [128,129]. Also, in man, the administration of allopurinol and oxipurinol leads to an increase in the specific activity of orotate phosphoribosyltransferase and orotidylic acid decarboxylase [129]. The enzymes were found to exist in a complex as three different molecular species with molecular weights of 55000, 80000 and 113 000 daltons. The larger forms of the complex were more stable than the smaller one. In the presence of allopurinol or oxipurinol ribonucleotides (but not the corresponding free bases) the largest, most stable species predominated [129]. 

There are several reports by Jones and co-workers dealing with the purification, properties and conformation of the orotate phosphoribosyltransferase and orotidylic acid decarboxylase enzyme complex present in mouse Ehrlich ascites cells [135-137]. Multiple molecular forms of orotidylic acid decarboxylase from human erythrocytes and human liver were studied by O Sullivan and co-workers [138,139]. A bifunctional enzyme complex of orotate phosphoribosyltransferase and orotidylic acid decarboxylase occurs also in mouse liver and brain [140], regardless of the developmental stage of the animal. Both enzyme activities remained co-ordinate in fetal, neonatal, immature and adult liver and brain. 

An inhibitory effect on orotidylic acid decarboxylase was also observed following 5-azacytidine [253,254], another highly active cytostatic agent [253-256]. The direct action of 5-azacytidine 5 -phosphate on enzyme activity in vitro has not yet been measured and the evidence for its interaction with the transformation of orotic acid came from the observation that 5-azacytidine increases its urinary excretion in mice [257,258]. The activity of orotidylic acid decarboxylase in liver extracts from 5-azacytidine-treated animals was also decreased in comparison to controls [258]. 

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. 

F21 Fox, R. M., Wood, M. H. and O Sullivan, W. J. Coordinate activity and lability of orotidylate phosphoribosyltransferase and decarboxylase in human erythrocytes, and the effects of allopurinol administration. J. Clin Invest., 50, 1050-1060 (1971)... 

Pyrazofurin in serum and urine. Because of the lack of analytical techniques, Sweeney, ad, (4) measured "PF-like activity" in the plasma of rats by determining the inhibition of orotidylic acid decarboxylase. This activity reached a peak level 6-8 hr after dosing (p.o. or i.m.) with substantial levels remaining in the plasma for 24 hr. Activity was detected for at least 3 days by 5 days all activity disappeared. This observed slow clearance... 

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