| Abstract |
Kinetic parameters of purine nucleoside phosphorylase (PNP) from Saccharomyces cerevisiae were measured. The Michaelis constants determined for substrates of the enzyme were 2.0 × 10^-4 M for inosine, 2.0 × 10^-3 M for deoxyinosine, 2.0 × 10^-5 M for guanosine and 2.0 10/sup -5/ M for deoxyguanosine. According to the ratio of relative K_cat/Km, 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 × 10^-4 M for formycin B as the competitive inhibitor of inosine, 9 × 10^-6 M for guanine as the competitive inhibitor of guanosine, 2 × 10^-4 M for hypoxanthine as the non competitive inhibitor of guanosine and 4.5 × 10^-4 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 2.0 × 10^-5 M, 2.6 × 10^-5 M and 8.5 × 10^-4 M, respectively, when inosine was the variable substrate. Guanosine and deoxyguanosine were also observed as competitive inhibitors with the Ki values of 1.8 × 10^-5 M and 3.0 × 10^-5 M, respectively, when deoxyinesine was the variable substrate. The results of alternative substrate sstudies suggested that a single enzyme acted on different nucleosides, inosine, deoxyinosine, adenosine, guanosine and deoxyguanosine. |
