GCase (glucosylceramidase) from Paenibacillus sp. TS12, a family 3 glycosidase, hydrolyses the β-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 β-D-glucopyranoside [Km = 0.27 ± 0.02 mM and k cat/Km = (2.1 ± 0.2) x 106 M -1·s-1] and 2,4-dinitrophenyl β-D- glucopyranoside [Km = 0.16 ± 0.02 mM and kcat/ Km = (2.9 ± 0.4) x 106 M-1·s -1] were used for continuous assay of the enzyme. The dependence of kcat (and kcat/Km) on pH revealed a dependence on a group of pKa ≤ 7.8 in the enzyme-substrate complex which must be protonated for catalysis. Incubation of GCase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucopyranoside caused time-dependent inactivation (Ki = 2.4 ± 0.7 mM and ki = 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 (> 104), 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.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology