Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247

A family 3 beta -glucosidase (EC 3.2.1.21) from Flavobacterium meningosepti cum has been cloned and overexpressed. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including subst rate reactivity, secondary kinetic isotope effects and inhibition studies. The stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacemen t reaction. Based on the k(cat) values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behaviour is consistent with a two-step mechanism involving the formation a nd breakdown of a glucosyl-enzyme intermediate. The large Bronsted constant (beta = -0.85) for the leaving-group-dependent portion (pK(a) of leaving p henols > 7) indicates substantial bond cleavage at the transition slate. Se condary deuterium kinetic isotope effects with 2,4-dinitrophenyl beta -D-gl ucopyanoside, o-nitrophenyl beta -3-D-glucopyranoside and p-cyanophenyl bet a -D-glucopyanoside as substrates were 1.17 +/- 0.02, 1.19 +/- 0.02 and 1.0 4 +/- 0.02 respectively. Theseresults support an S(N)1-like mechanism for t he deglucosylation step and an S(N)2-like mechanism for the glucosylation s tep. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (k(cat)/K-m) of the D247G and D247N mutants w ere 30000- and 200000-fold lower respectively than that of the wild-type en zyme, whereas the D247E mutant retained 20 % of wild-type activity. These r esults indicate that Asp-247 is an essential amino acid. It is likely that this residue functions as a nucleophile in the reaction. This conclusion is supported by the kinetics of the irreversible inactivation of the wild-typ e enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mu tant.