Electrostatic control of enzyme reactions: the mechanism of inhibition of glucose oxidase by putrescine

Abstract

The interaction of putrescine dihydrochloride with glucose oxidase is reported. At pH 7.65 glucose oxidase is strongly anionic (Z = -80). The pKa of an essential acidic group on the reduced form of the enzyme is extremely sensitive to ionic strength, as predicted by simple electrostatic theory [J. G. Voet, J. Coe, J. Epstein, V. Matossian, and T. Shipley (1981) Biochemistry 20, 7182-7185]. Putrescine dihydrochloride was found to inhibit glucose oxidase at pH 7.65 at a constant ionic strength of 0.05. The kinetics do not obey simple competitive inhibition, however. The data can best be explained by a model in which change in the electrostatic potential of the enzyme on putrescine binding changes the observed pKa of the essential acidic group. The pH dependence of putrescine inhibition supports this interpretation. At I = 0.05, 5 mM putrescine was found to change the pKa of the essential acidic group from 7.6 to 7.1. The shift in the pKa as a function of putrescine concentration at pH 7.7 and I = 0.05 also supports the model presented. The Ka for putrescine to the active form of the enzyme was calculated to be 4.2 mM.