title: Substrate-induced inactivation of a crippled beta-glucosidase mutant: identification of the labeled amino acid and mutagenic analysis of its role.
abstract: The beta-glucosidase from Agrobacterium sp. catalyzes the hydrolysis of beta-glucosides via a covalent alpha-D-glucopyranosyl-enzyme intermediate involving Glu358. Hydrolysis of 2,4-dinitrophenyl beta-D-glucopyranoside by the low activity Glu358Asp mutant of Agrobacterium beta-glucosidase is accompanied by time-dependent inactivation of the enzyme. Through kinetic studies, labeling, and sequence analysis, inactivation is shown to be a consequence of the occasional (1 time in 1100) attack of Tyr298 on the anomeric center of the substrate, in place of the catalytic nucleophile, with formation of a stable alpha-D-glucopyranosyl tyrosine residue. Tyr298 is conserved throughout family 1 of glycoside hydrolases, an indication of a possible role in catalysis. Results of a kinetic analysis of the Tyr298Phe mutant are consistent with a function of Tyr298 in both orienting the nearby nucleophile Glu358 and stabilizing its deprotonated state in the free enzyme.
authors.affiliation: Protein Engineering Network of Centres of Excellence of Canada, Vancouver, British Columbia, Canada.
authors.forename: J C
authors.initials: JC
authors.lastname: Gebler
authors.forename: D E
authors.initials: DE
authors.lastname: Trimbur
authors.forename: A J
authors.initials: AJ
authors.lastname: Warren
authors.forename: R
authors.initials: R
authors.lastname: Aebersold
authors.forename: M
authors.initials: M
authors.lastname: Namchuk
authors.forename: S G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012231:Rhizobium / Q000201:enzymology*; D017421:Sequence Analysis; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001617:beta-Glucosidase