Temperature-dependent change in the rate-limiting step of beta-glucosidase catalysis.

Abstract

When beta-glucosidase from sweet almonds reacts with p-nitrophenyl-beta-D-glucoside at subzero temperatures, a rapid release of p-nitrophenol, stoichiometric with enzyme concentration, occurs prior to turnover. This observation is interpreted as reflecting rapid formation of a glucose-enzyme intermediate, followed by rate-limiting breakdown. The energies of activation for the formation and breakdown of the intermediate were 6.0 +/- 2 and 19.0 +/- 2 kcal mol-1, respectively. Extrapolation of the reaction rates to higher temperatures suggests an isokinetic temperature circa 24 degrees C. Measurement of kcat in aqueous solution as a function of temperature yields a breakpoint in the Arrhenius plot at 20 degrees C and activation energies above and below this point which correspond to the values at subzero temperatures (6.7 and 17.0 kcal mol-1, respectively). This breakpoint thus reflects a change in the rate-determining step from formation of the intermediate at higher temperatures to breakdown at lower values, and explains why a "burst" of p-nitrophenol is observed at low, but not high, temperatures with this substrate. The significance of this finding to other glycohydrolases, and enzyme catalysis in general, is considered.