Purine nucleotide pyrophosphorylases,

Davidson, J. D., Bradley, T. R., Roosa, R. A., and Law, L. W. (1962), Purine nucleotide pyrophosphorylases in 8-azaguanine-sensitive and -resistant P388 leukemias, J. Natl. Cancer Inst. 29, 789-803. 

F2. Flaks, J. G., Erwin, M. J., and Buchaium, J. M., Biosynthesis of the purines— the synthesis of adenosine 5 phosphate and 5-amino-4-imidazolecarboxamide ribotide by a nucleotide pyrophosphorylase. J. Biol. Chem. 288, 201 (1957). 

It was not until 1953 that Goldwasser (9) and Williams and Buchanan (10) showed that purine bases could be converted to ribonucleotides by a one-step process, without the intermediate formation of ribonucleosides. The source of the ribose phosphate moiety was discovered in 1955 to be PP-ribose-P in the course of studies of adenylate synthesis by Kornberg et al. 11), and of inosinate synthesis by Korn et al. 12) extracts of yeast, beef liver, and pigeon liver were employed. The enzymes involved were at first called nucleotide pyrophosphorylases, but are now known as purine phosphoribosyltransferases. The general reaction is... 

The carbamyl phosphate is condensed with a molecule of aspartate giving ureidosuccinic acid, from which orotic acid is formed by cyclization and oxidation. In the presence of PRPP and a pyrophosphorylase this acid forms a ribotide and decarboxylation yields uridine monophosphate. The decarboxylation of the product of amination of orotidine phosphate gives cytidine monophosphate (Fig. 75). It can be seen that the pentose intermediate in pyrimidine nucleotide biosynthesis is PRPP, the same as for purine nucleotide biosynthesis. 

The direct synthesis of nucleotides can take place by the reaction of a purine or pyrimidine base and PRPP according to reaction (14). The reaction is catalyzed by nucleotide pyrophosphorylase. 

Two separate purine nucleotide p3rrophosphorylases have been described (61). One enzyme converts hypoxanthine, guanine (61, 68), and 6-mercaptopurine (63) to their ribotides in the presence of PRPP, while a second enzyme is active with adenine and 4-amino-5-imidazolecarboxa-mide (64). The latter enzyme has been purified from beef liver. Nucleotide pyrophosphorylase has also been purified from yeast (61). 

Amino-4-imidazole carboxamide ribotide, a precursor only two steps removed (formylation and cycli-zation) from inosinic acid, can be synthesized by the direct condensation of the imidazole with 5-phosphori-bosyl pyrophosphate. The enzyme catalyzing this reaction was purified from an acetone powder of beef liver. The same enzyme (AMP pyrophosphorylase) catalyzes the condensation of adenine, guanine, and hypoxan-thine. Nucleoside phosphorylase is an enzyme that catalyzes the formation of a ribose nucleoside from a purine base and ribose-1-phosphate. Guanine, adenine, xanthine, hypoxanthine, 2,6-diaminopurine, and aminoimidazole carboxamide are known to be converted to their respective nucleosides by such a mechanism. In the presence of a specific kinase and ATP, the nucleoside is then phosphorylated to the corresponding nucleotide. 

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. 

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. 

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