title: Trapping covalent intermediates on beta-glycosidases.
abstract: The mechanism-based inactivation and subsequent identification of the nucleophilic residue using mass spectrometry have been successfully applied and used to identify the active-site nucleophile in numerous beta-glycosidases, as illustrated using C. fimi exoglycanase. Evidence for a covalent glycosyl-enzyme intermediate has come from X-ray crystallographic analysis of trapped complexes, the first being that of the trapped fluoroglycosyl-enzyme intermediate of Cex. The crystal structure of the trapped fluorocellobiosyl-enzyme complex for Cex has provided useful insights into catalysis and the roles of specific residues at the active site. In addition, information about the conformation of the natural sugar in the covalently bound state and the interactions at the active site was obtained using a mutant form of Cex.
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Wicki
authors.forename: David R
authors.initials: DR
authors.lastname: Rose
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D018360:Crystallography, X-Ray; D043364:Endo-1,4-beta Xylanases; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005459:Fluorides / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002241:Carbohydrates; D005456:Fluorescent Dyes; D006026:Glycoside Hydrolases; D014995:Xylosidases; D001617:beta-Glucosidase; D043364:Endo-1,4-beta Xylanases; C090001:XynB xylanase; D005459:Fluorides