Mechanism of the family 1,beta-glucosidase from Streptomyces sp: Catalyticresidues and kinetic studies

The Streptomyces sp. beta -glucosidase (Bg13) is a retaining glycosidase th at belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-ni trophenyl beta -D-glycosides revealed that the highest k(cat)/K-M values ar e obtained with glucoside (with strong substrate inhibition) and fucoside ( with no substrate inhibition) substrates and that Bg13 has 10-fold glucosid ase over galactosidase activity. Reactivity studies by means of a Hammett a nalysis using a series of substituted aryl beta -glucosides gave a biphasic plot log k(cat) vs pK(a) of the phenol aglycon: a linear region with a slo pe of beta (lg) = -0.8 for the less reactive substrates (pK(a) > 8) and no significant dependence for activated substrates (pK(a) 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glyco syl-enzyme intermediate is rate limiting for the former substrates, while h ydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family I beta -gl ucosidases, glutamic acids 178 and 383 were changed to glutamine and alanin e by site-directed mutagenesis. Mutation of Glu 178 to Gin and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (10(5)-10(6)-fold) were obtained for mutants at Gl u383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exo genous nucleophile. The E178A mutant yielded the beta -glucosyl azide adduc t (by H-1 NMR) with a 200-fold increase on k(cat) for the 2,4-dinitrophenyl glucoside but constant k(cat)/K-M on azide concentration. On the other han d, the E383A mutant with the same substrate gave the alpha -glucosyl azide product and a 100-fold increase in k(cat) at 1 M azide. In conclusion, Glu1 78 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bg13 beta -glucosidase displays ki netic and mechanistic properties similar to other family I enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.