MULTIPLE BINDING-SITES FOR TETRAHEDRAL OXYANION INHIBITORS OF BOVINE SPLEEN PURPLE ACID-PHOSPHATASE

The theory of multiple inhibition kinetics has been extended to enzyme s for which one inhibitor is noncompetitive and the other exhibits mix ed inhibition. Plots of reciprocal velocity versus the concentration o f either inhibitor at various fixed concentrations of the second inhib itor are predicted to give parallel lines if binding of the inhibitors is mutually exclusive and intersecting lines if the inhibitors intera ct at different sites on the enzyme. Application of this analysis to t he purple acid phosphatase from bovine spleen in the presence of molyb date (a noncompetitive inhibitor) and phosphate (which exhibits mixed inhibition) results in parallel lines in the reciprocal velocity plots , indicating that phosphate and molybdate compete for a common site; s ince molybdate is a noncompetitive inhibitor, this site is inferred to be distinct from the site at which substrate binds and is hydrolyzed. Extension of these ideas suggests that phosphate ester substrates sho uld be capable of binding to the molybdate-binding site as well as to the active site, and evidence for substrate inhibition at high substra te concentrations has been obtained. The implications of these finding s for interpretation of previous spectroscopic studies of purple acid phosphatase complexes with tetrahedral oxyanions are discussed.