Mechanism of the family 1 β-glucosidase from Streptomyces sp: Catalytic residues and kinetic studies

The Streptomyces sp. β-glucosidase (Bg13) is a retaining glycosidase that belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-nitrophenyl β-D-glycosides revealed that the highest kcat/KM values are obtained with glucoside (with strong substrate inhibition) and fucoside (with no substrate inhibition) substrates and that Bg13 has 10-fold glucosidase over galactosidase activity. Reactivity studies by means of a Hammett analysis using a series of substituted aryl β-glucosides gave a biphasic plot log kcat vs pKa of the phenol aglycon: a linear region with a slope of β1g = -0.8 for the less reactive substrates (pKa > 8) and no significant dependence for activated substrates (pKa < 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glycosyl-enzyme intermediate is rate limiting for the former substrates, while hydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family 1 β-glucosidases, glutamic acids 178 and 383 were changed to glutamine and alanine by site-directed mutagenesis. Mutation of Glu178 to Gln and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (105-106-fold) were obtained for mutants at Glu383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exogenous nucleophile. The E178A mutant yielded the β-glucosyl azide adduct (by 1H NMR) with a 200-fold increase on kcat for the 2,4-dinitrophenyl glucoside but constant kcat/KM on azide concentration. On the other hand, the E383A mutant with the same substrate gave the α-glucosyl azide product and a 100-fold increase in kcat at 1 M azide. In conclusion, Glu178 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bg13 β-glucosidase displays kinetic and mechanistic properties similar to other family 1 enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.