Purine nucleoside phosphorylase. Kinetic mechanism of the enzyme from calf spleen.
Ribose 1-phosphate, phosphate, and acyclovir diphosphate quenched the fluorescence of purine nucleoside phosphorylase at pH 7.1 and 25 degrees C. The fluorescence of enzyme-bound guanine was similar to that of anionic guanine in ethanol. Guanine and ribose 1-phosphate bound to free enzyme, whereas inosine and guanosine were not bound to free enzyme in the absence of phosphate. Thus, synthesis proceeded by a random mechanism, and phosphorolysis proceeded by an ordered mechanism. Steady-state kinetic data for the phosphorolysis of 100 microM guanosine were fitted to a bifunctional kinetic model with catalytic rate constants of 22 and 1.3 s-1. The dissociation rate constants for guanine from the enzyme-guanine complex at high and low phosphate concentrations were similar to the catalytic rate constants. Fluorescence changes of the enzyme during phosphorolysis suggested that ribose 1-phosphate dissociated from the enzyme ribose 1-phosphate-guanine complex rapidly and that guanine dissociated from the enzyme-guanine complex slowly. The association and dissociation rate constants for acyclovir diphosphate, a potent inhibitor of the enzyme (Tuttle, J. V., and Krenitsky, T. A. (1984) J. Biol. Chem. 259, 4065-4069), were also dependent on phosphate concentration. The effects of phosphate are discussed in terms of a dual functional binding site for phosphate.
Porter
D J
DJ
Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
D000212:Acyclovir / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D005453:Fluorescence; D006147:Guanine / Q000378:metabolism; D006151:Guanosine / Q000494:pharmacology; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism; D007288:Inosine / Q000494:pharmacology; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000494:pharmacology; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity
D007042:Hypoxanthines; D009943:Organophosphorus Compounds; D012267:Ribosemonophosphates; D006151:Guanosine; C031154:ribose 1-phosphate; D019271:Hypoxanthine; D007288:Inosine; D006147:Guanine; D011683:Purine-Nucleoside Phosphorylase; D000212:Acyclovir