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Kinetic parameters of purine nucleoside phosphorylase (PNP) from Saccharomyces cerevisiae were measured. The Michaelis constants determined for substrates of the enzyme were 2.OX 10-´ M for inosine, 2.OX 10-´ M for deoxyinosine, 2.OX 10_´ M for guanosine and 2.OX 10_1 M for deoxyguanosine. According to the ratio of relative K JKm, substrate specificity of each nucleoside was in the order of guanosine or deoxyguanosine, inosine and deoxyinosine. Cosubstrate, phosphate, revealed downward curvature in lineweaver-Burk plot at high concentrations, indicating a negative cooperativity between subunits. The inhibition constants for purine analogs were measured to be 6 X 10-´ M for formycin B as the competitive inhibitor of inosine, 9 X 10-` M for guanine as the competitive inhibitor of guanosine, 2X 10-´ M for hypoxanthine as the non competitive inhibitor of guanosine and 4.5X 10-´ M for 6-mercaptopurine as the non competitive inhibitor of guanosine. Alternative substrates, guanosine, deoxyguanosine and adenosine were found to act as competitive inhibitors with Ki values of 2.OX 10-s M, 2.6X 10-´ M and 8.5 X 10-´ M, respectively, when inosine was the variable substrate. Guanosine and deoxyguanosine were also observed as competitive inhibitors with the Ki values of 1.8X 10_, M and 3.OX 10_1 M, respectively, when deoxyinosine was the variable substrate. The results of alternative substrate studies suggested that a single enzyme acted on different nucleosides, inosine, deoxyinosine, adenosine, guanosine and deoxyguanosine.
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