Kinetics of the hydrolysis of cellobiose catalysed by β-glucosidase

Abstract

The hydrolysis of cellobiose catalysed by β-glucosidase has been investigated by experimental techniques which allow the course of reaction to be followed continuously. They involve assaying the product glucose by the use of ATP, hexokinase, glucose-6-phosphate dehydrogenase, and nicotinamide adenine dinucleotide phosphate (NADP); the latter is converted into its reduced form NADPH which absorbs strongly at 340 nm. Rates were measured at nine pH values varying from 5 to 6.9, at substrate concentrations varying from 0.2 to 3.2 mM, and at temperatures varying from 10 to 37 °C. The pH dependence revealed pK values of 4.9 and 6.5 in the free enzyme at 24 °C, and these are little changed on complex formation. The rates measured over a range of temperature, as interpreted by Arrhenius plots, revealed an inflexion at 23 °C, found consistently under all conditions. The results are analyzed in terms of the mechanism[Formula: see text]It was found possible to obtain, for the four elementary reactions, activation energies and entropies of activation which explain the inflexion at 23 °C and the Arrhenius behavior above and below that temperature. Profiles are constructed showing the variations of entropy and enthalpy during the course of an individual reaction.