Kinetic properties and inhibition of orotidine 5'-phosphate decarboxylase. Effects of some allopurinol metabolites on the enzyme.

The land 7-ribosyl 5’-phosphates of oxipurinol [4,6dihydroxypyrazolo(3,4 d)pyrimidine] were enzymatically synthesized. These nucleotides, 3-xanthosine S’-phosphate, and the 1-ribosyl 5’-phosphate of allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] were characterized by spectral, chromatographic, and enzymatic procedures. The oxipurinol nucleotides were found to be potent competitive inhibitors of orotidine 5’-phosphate decarboxylase from yeast. This enzyme exhibited bimodal substrate saturation kinetics, the characteristics of which were consistent with the involvement of a single enzyme. Inhibition by nucleotides was also bimodal; thus, two K, values could be determined for each inhibitor. The most effective inhibitor tested was 1-ribosyloxipurinol 5’-phosphate, which had Ki values of 0.02 and 0.003 PM at high (12 to 48 PM) and low (0.5 to 2 PM) substrate concentrations, respectively. The corresponding N-7 derivative had corresponding Ki values of 0.7 and 0.06 PM. l-Ribosylallopurinol 5’-phosphate, 3xanthosine 5’-phosphate, and several naturally occurring nucleotides were less inhibitory. Nucleotide inhibitors also stabilized the enzyme against loss of activity at 37”. Inhibition constants determined with the enzyme from rat liver were similar to those from yeast. The findings furnish direct, quantitative support for the idea that it is the inhibition of orotidine 5’-phosphate decarboxylase by the nucleotides of oxipurinol that is primarily responsible for the increased urinary excretion of erotic acid and orotidine in patients treated with allopurinol.