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 kcat/Km=(2.1±0.2)×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)×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.
Paenibacillus sp. TS12 glucosylceramidase: kinetic studies of a novel sub-family of family 3 glycosidases and identification of the catalytic residues
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Krisztina PAAL, Makoto ITO, Stephen G. WITHERS; Paenibacillus sp. TS12 glucosylceramidase: kinetic studies of a novel sub-family of family 3 glycosidases and identification of the catalytic residues. Biochem J 15 February 2004; 378 (1): 141–149. doi: https://doi.org/10.1042/bj20031028
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