Orotidylic acid decarboxylase

Pyrazomycin. Pyrazomycin (11), 3-(l-p-D-ribofuranosyl)-4-hydroxypyrazole 5-carboxamide, is isolated from S. Candidas (1—4,9,10). The incorporation of [2-13C]acetate and [1- and U-14C]glutamate into the four contiguous carbons of pyrazomycin has been reported (11,12). Pyrazomycin 5 -phosphate inhibits orotidylic acid decarboxylase. Pyrazomycin inhibits adenosine phosphorylation and decreases the incorporation of deoxyuridine into DNA of Novikoff hepatoma cells in culture. It also inhibits the growth of tumor cells and the cytopathic effects of vaccinia, herpes simplex, vesicular stomatitis, Newcasde disease, measles, Sindbis, polio, hepatitis A, and coxsackie viruses (13,14). The inhibitory action of (11) on viral multiplication is reversed by uridine. 

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]. 

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 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. 

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]. 

Enhanced excretion of orotic acid was observed under different physiological [209,210] and nutritional [211-217] conditions. The amount of orotic acid excreted during human pregnancy is about 20-40 mg per day and does not vary substantially during the course of pregnancy [209,210]. Inherited deficiencies of the urea cycle [218], purine nucleoside phosphorylase [219], and especially of orotate phosphoribosyltransferase and orotidylic acid decarboxylase also result in an increased excretion of orotic acid. 

Orotidylic acid decarboxylase from homozygous mutant cells was found to be more thermostabile and exhibited different electrophoretic mobility when compared to the enzyme from normal cells [243]. Although the differences have been shown for this enzyme only, they could reflect alterations in the primary structure of either orotidylic acid decarboxylase... 

The first known drug affecting the orotate pathway was 6-azauridine [245,246], This analogue is phosphorylated to 6-azauridine 5 -monophosphate which acts as a competitive inhibitor of orotidylic acid decarboxylase [247], Therapeutic use of 6-azauridine [248,249] is occasionally complicated by a pronounced crystalluria. Owing to the block of orotidylic acid decarboxylase, large amounts of orotidine and orotic acid are excreted in urine. After the infusion of 6-azauridine the excretion of orotic acid precedes orotidine and the former disappears more rapidly from the urine. Psoriatic patients on azaribine (triacetylated form of 6-azauridine given orally) excreted 0.2-1.3 g of orotic acid and orotidine per day [250]. 

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. 

Fluoro-orotic acid undergoes in the liver the same conversion as orotic acid [269,270]. The 5-fluoro analogue serves as a substrate for orotate phosphoribosyltransferase [270] and the anomalous nucleoside 5 -phosphate so produced inhibits orotidylic acid decarboxylase. A number of... 

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)... 

Uridylic acid, the nucleotide found in RNA, is formed by decarboxylation of orotidylic acid in the presence of orotidine-5 -phosphate decarboxylase, an enzyme purified from yeast. This is an irreversible reaction that has been observed in bacteria, birds, and several mammalian tissues. The antimetabolite 6-azauracil blocks orotidylic acid decarboxylase. 

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]. 

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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