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). 

Fourth, the orotate ring is transferred to phosphoribosyl pyrophosphate (PRPP) to form a 5 ribose-phosphate, orotidylic acid. 

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. 

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. 

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... 

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). 

This enzyme catalyses the reaction which synthesises orotidylic acid, the precursor of UMP. It may be assayed radiochemically [523]. 

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... 

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