Mechanism, mutagenesis, and chemical rescue of beta-mannosidase from Cellulomonas fimi

The chemical mechanism of a retaining beta-mannosidase from Cellulomonas fimi has been characterized through steady-state kinetic analyses with a range of substrates, coupled with chemical rescue studies on both the wild-type enzyme and mutants in which active site carboxyl groups have been replaced. Studies with a series of aryl beta-mannosides of vastly different reactivities (pK(a)(1g) = 4-10) allowed kinetic isolation of the glycosylation and deglycosylation steps. Substrate inhibition was observed for all but the least reactive of these substrates. Bronsted analysis of k(cat) revealed a downward breaking plot (beta(1g) = -0.54 +/- 0.05) that is consistent with a change in rate-determining step (glycosylation to deglycosylation), and this was confirmed by partitioning studies with ethylene glycol. The pH dependence of k(cat)/K-m follows an apparent single ionization of a group of pK(a) = 7.65 that must be protonated for catalysis. The tentative assignment of E429 as the acid-base catalyst of Man2A on the basis of sequence alignments with other family 2 glycosidases was confirmed by the increased turnover rate observed for the mutant E429A in the presence of azide and fluoride, leading to the production of beta-mannosyl azide and beta-mannosyl fluoride, respectively. A pH-dependent chemical rescue of E429A activity is also observed with citrate. Substantial oxocarbenium ion character at the transition state was demonstrated by the alpha-deuterium kinetic isotope effect for Man2A E429A of alpha-D-(V) = 1.12 +/- 0.01. Surprisingly, this isotope effect was substantially greater in the presence of azide (alpha-D-(V) = 1.166 +/- 0.009). Likely involvement of acid/base catalysis was revealed by the pH dependence of kcat for Man2A E429A, which follows a bell-shaped profile described by pKa values of 6.1 and 8.4, substantially different from that of the wild-type enzyme. The glycosidic bond cleaving activity of Man2A E519A and E519S nucleophile mutants is restored with azide and fluoride and appears to correlate with the corresponding ``glycosynthase{''} activities. The contribution of the substrate 2-hydroxyl to stabilization of the Man2A glycosylation transition state (DeltaDeltaG(double dagger) = 5.1 kcal mol(-1)) was probed using a 2-deoxymannose substrate. This value, surprisingly, is comparable to that found from equivalent studies with beta-glucosidases despite the geometric differences at C-2 and the importance of hydrogen bonding at that position. Modes of stabilizing the mannosidase transition state are discussed.