Mechanism, Mutagenesis, and Chemical Rescue of a β-Mannosidase from Cellulomonas fimi†

The chemical mechanism of a retaining β-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 β-mannosides of vastly different reactivities (pKalg = 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 kcat revealed a downward breaking plot (βlg = −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 kcat/Km follows an apparent single ionization of a group of pKa = 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 al...