title: Glucosyl epi-cyclophellitol allows mechanism-based inactivation and structural analysis of human pancreatic α-amylase.
abstract: As part of a search for selective, mechanism-based covalent inhibitors of human pancreatic α-amylase we describe the chemoenzymatic synthesis of the disaccharide analog α-glucosyl epi-cyclophellitol, demonstrate its stoichiometric reaction with human pancreatic α-amylase and evaluate the time dependence of its inhibition. X-ray crystallographic analysis of the covalent derivative so formed confirms its reaction at the active site with formation of a covalent bond to the catalytic nucleophile D197. The structure illuminates the interactions with the active site and confirms OH4' on the nonreducing end sugar as a good site for attachment of fluorescent tags in generating probes for localization and quantitation of amylase in vivo.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Sami
authors.initials: S
authors.lastname: Caner
authors.affiliation: Department of Chemistry, Faculty of Science, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Leiden Institute of Chemistry, Leiden University, The Netherlands.
authors.forename: Jianbing
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Department of Chemistry, Faculty of Science, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Nham T
authors.initials: NT
authors.lastname: Nguyen
authors.affiliation: Leiden Institute of Chemistry, Leiden University, The Netherlands.
authors.forename: Hermen
authors.initials: H
authors.lastname: Overkleeft
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Brayer
authors.affiliation: Department of Chemistry, Faculty of Science, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D020134:Catalytic Domain; D003198:Computer Simulation; D003511:Cyclohexanols / Q000494:pharmacology*; D004187:Disaccharides / Q000494:pharmacology*; D004852:Epoxy Compounds / Q000494:pharmacology*; D006801:Humans; D006860:Hydrogen Bonding; D007294:Inositol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D055292:Pancreatic alpha-Amylases / Q000737:chemistry* / Q000378:metabolism*; D014867:Water; D014961:X-Ray Diffraction
chemical_list: D003511:Cyclohexanols; D004187:Disaccharides; D004852:Epoxy Compounds; C000619073:glucosyl cyclophellitol; D014867:Water; C063285:cyclophellitol; D007294:Inositol; D055292:Pancreatic alpha-Amylases; C011423:conduritol epoxide
keywords: GH13 structure; activity-based probes; amylase; conduritol epoxide; glycosyl enzyme; mechanism-based inhibition