The family 1 beta-glucosidases from Pyrococcus furiosus and Agrobacterium faecalis share a common catalytic mechanism

Comparisons of catalytic mechanisms have not previously been performed for homologous enzymes from hyperthermophilic and mesophilic sources. Here, the beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus w as recombinantly produced in Escherichia coli and shown to have biophyscial and biochemical properties identical to those of the wild-type enzyme. Mor eover, the recombinant enzyme was subjected to a detailed kinetic investiga tion at 95 degrees C to compare its catalytic mechanism to that determined at 37 degrees C for the beta-glucosidase (abg) from the mesophilic bacteriu m, Agrobacterium faecalis [Kempton, J., and Withers, S. G. (1992) Biochemis try 31, 9961]. These enzymes have amino acid sequences that are 33% identic al and have been classified as family 1 glycosyl hydrolases on the basis of amino acid sequence similarities. Both enzymes have similar broad specific ities for both sugar and aglycone moieties and exhibit nearly identical pH dependences for their kinetic parameters with several different substrates. Bronsted plots were constructed for bgl at several temperatures using a se ries of aryl glucoside substrates. These plots were concave downward at all temperatures, indicating that bgl utilized a two-step mechanism similar to that of abg and that the rate-limiting step in this mechanism did not chan ge with temperature for any given aryl glucoside. The Bronsted coefficient for bgl at 95 degrees C (beta(1g) = -0.7) was identical to that for abg at 37 degrees C and implies that these enzymes utilize nearly identical transi tion states, at least in regard to charge accumulation on the departing gly cosidic oxygen. In addition, a high correlation coefficient (rho = 0.97) fo r the linear free energy relationship between these two enzymes and similar inhibition constants for these two enzymes with several ground state and t ransition state analogue inhibitors further indicate that these enzymes sta bilize similar transition states. The mechanistic similarities between thes e two enzymes are noteworthy in light of the large difference in their temp erature optima. This suggests that, in the presumed evolution that occurred between the hyperthermophilic archaeal enzyme and the mesophilic bacterial enzyme, structural modifications must have been selected which maintained the integrity of the active site structure and, therefore, the specificity of transition state interactions, while adapting the overall protein struct ure to permit function at the appropriate temperature.