PROBING THE MECHANISM OF BACILLUS 1,3-1,4-BETA-D-GLUCAN 4-GLUCANOHYDROLASES BY CHEMICAL RESCUE OF INACTIVE MUTANTS AT CATALYTICALLY ESSENTIAL RESIDUES

The role of the key catalytic residues Glu134 and Glu138 in the retain ing 1,3-1,4-beta-glucanase from Bacillus licheniformis is probed by a chemical rescue methodology based on enzyme activation of inactive mut ants by the action of added nucleophiles. While Glu134 was proposed as the catalytic nucleophile on the basis of affinity labeling experimen ts, no functional proof supported the assignment of Glu138 as the gene ral acid-base catalyst. Alanine replacements are prepared by site-dire cted mutagenesis to produce the inactive E138A and E134A mutants. Addi tion of azide reactivates the mutants in a concentration-dependent man ner using an activated 2,4-dinitrophenyl glycoside substrate. The chem ical rescue operates by a different mechanism depending on the mutant as deduced from H-1 NMR monitoring and kinetic analysis of enzyme reac tivation. E138A yields the beta-glycosyl azide product arising from nu cleophilic attack of azide on the glycosyl-enzyme intermediate, thus p roving that Glu138 is the general acid-base residue. Azide activates t he deglycosylation step (increasing k(cat)), but it also has a large e ffect on a previous step (as seen by the large decrease in K-M, the in crease in k(cat)/K-M, and the pH dependence of activation), probably i ncreasing the rate of glycosylation through Bronsted acid catalysis by enzyme-bound HN3. By contrast, azide reactivates the E134A mutant thr ough a single inverting displacement to give the alpha-glycosyl azide product, consistent with Glu134 being the catalytic nucleophile. Forma te as an exogenous nucleophile has no effect on the E138A mutant, wher eas it is a better activator of E134A than azide. Although the reactio n yields the normal hydrolysis product, a transient compound was detec ted by H-1 NMR, tentatively assigned to the alpha-glycosyl formate add uct. This is the first case where a nonmodified sugar gives a long-liv ed covalent intermediate that mimics the proposed glycosyl-enzyme inte rmediate of retaining glycosidases.