title: Paenibacillus sp. TS12 glucosylceramidase: kinetic studies of a novel sub-family of family 3 glycosidases and identification of the catalytic residues.
abstract: 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 kcat/K(m)=(2.1+/-0.2)x10(6) M(-1) x 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)x10(6) M(-1) x s(-1)] were used for continuous assay of the enzyme. The dependence of kcat (and kcat/K(m)) on pH revealed a dependence on a group of pK(a)< or =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-D-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.
authors.affiliation: Protein Engineering Network of Centres of Excellence of Canada and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
authors.forename: Krisztina
authors.initials: K
authors.lastname: Paal
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D005962:Glucosylceramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000145:classification; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D019810:Sodium Azide / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D019810:Sodium Azide; D006026:Glycoside Hydrolases; D005962:Glucosylceramidase