MECHANISTIC CONSEQUENCES OF MUTATION OF ACTIVE SITE CARBOXYLATES IN A RETAINING BETA-1,4-GLYCANASE FROM CELLULOMONAS FINI

摘要

The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate. The roles of three conserved active site carboxylic acids in this enzyme have been probed by detailed kinetic analysis of mutants modified at these three positions. Elimination of the catalytic nucleophile (E233A) results in an essentially inactive enzyme, consistent with the important role of this residue. However addition of small anions such as azide or formate restores activity, but as an inverting enzyme since the product formed under these conditions is the cr-glycosyl azide, Shortening of the catalytic nucleophile (E233D) reduces the rates of both formation and hydrolysis of the glycosyl-enzyme intermediate some 3000-4000-fold. Elimination of the acid/base catalyst (E127A) yields a mutant for which the deglycosylation step is slowed some 200-300-fold as a consequence of removal of general base catalysis, but with little effect on the transition state structure at the anomeric center, Effects on the glycosylation step due to removal of the acid catalyst depend on the aglycon leaving group ability, with minimal effects on substrates requiring no general acid catalysis but large (>10(5)-fold) effects on substrates with poor leaving groups. The Bronsted beta(1g) value for hydrolysis of aryl cellobiosides was much larger (beta(1g) similar to -1) for the mutant than for the wild-type enzyme (beta(1g) = -0.3), consistent with removal of protonic assistance. The pH-dependence was also significantly perturbed. Mutation of a third conserved active site carboxylic acid (E123A) resulted in rate reductions of up to 1500-fold on poorer substrates, which could be largely restored by addition of azide, but without the formation of glycosyl azide products. These results suggest a simple strategy for the identification of the key active site nucleophile and acid/base catalyst residues in glycosidases without resort to active site labeling.