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 p K a 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 p K a 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 p K a of the essential acidic group from 7.6 to 7.1. The shift in the p K a as a function of putrescine concentration at pH 7.7 and I = 0.05 also supports the model presented. The K a for putrescine to the active form of the enzyme was calculated to be 4.2 m m.