Kinetics of the hydrolysis of cellobiose catalysed by β-glucosidase

Abstract

The hydrolysis of cellobiose catalyzed by .beta.-glucosidase was 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 NADP; the latter is converted into its reduced form NADPH which absorbs strongly at 340 nm. Rates were measured at 9 pH values varying from 5 to 6.9, at substrate concentrations varying from 0.2-3.2 mM, and at temperatures varying from 10-37.degree. C. The pH dependence revealed pK values of 4.9 and 6.5 in the free enzyme at 24.degree. 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.degree. C, found consistently under all conditions. The results were analyzed in terms of the mechanism: .**GRAPHIC**. It was found possible to obtain, for the 4 elementary reactions, activation energies and entropies of activation which explain the inflexion at 23.degree. C and the Arrhenius behavior above and below that temperature. Profiles were constructed showing the variations of entropy and enthalpy during the course of an individual reaction.