Paenibacillus sp TS12 glucosylceramidase: kinetic studies of a novel sub-family of family 3 glycosidases and identification of the catalytic residues

GCase (glucosylceramidase) from Paenibacillus sp. TS12, a family 3 glycosidase, hydrolyses the beta-glycosidic linkage of glucosylceramide with retention of anomeric configuration via a two-step, double-displacement mechanism. Two carboxyl residues are essential for catalysis, one functioning as a nucleophile and the other as a general acid/base catalyst. p-Nitrophenyl beta-D-glucopyranoside {[}K-m = 0.27 +/- 0.02 mM and k(cat)/K-m = (2.1 +/- 0.2) x 10(6) M-1 (.) s(-1)] and 2,4-dinitrophenyl beta-D-glucopyranoside {[}K-m = 0.16 +/- 0.02 mM and k(cat)/K-m = (2.9 +/- 0.4) x 10(6) M-1 (.) s(-1)] were used for continuous assay of the enzyme. The dependence of k(cat) (and k(cat)/K-m) on pH revealed a dependence on a group of pK(a) less than or equal to 7.8 in the enzyme-substrate complex which must be protonated for catalysis. Incubation of GCase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-glucopyranoside caused time-dependent inactivation (K-i = 2.4 +/- 0.7 mM and k(i) = 0.59 +/- 0.05 min(-1)) due to the accumulation of a trapped glycosyl-enzyme intermediate. Electrospray ionization MS analysis of the peptic digest of this complex showed that the enzyme was covalently labelled by the reagent at Asp-223, consistent with its role as nucleophile. A mutant modified at this residue (D223G) showed substantially reduced activity compared with the wild type (> 10(4)), but this activity could be partially restored by addition of formate as an external nucleophile. Kinetic analysis of the mutant E411A indicated that Glu-411 serves as the general acid/base catalytic residue since this mutant was pH-independent and since considerable GCase activity was restored upon addition of azide to E411A, along with formation of a glycosyl azide product.