title: Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases.
abstract: The enzymatic mechanism by which retaining glycosyltransferases (GTs) transfer monosaccharides with net retention of the anomeric configuration has, so far, resisted elucidation. Here, direct detection of covalent glycosyl-enzyme intermediates for mutants of two model retaining GTs, the human blood group synthesizing α-(1 → 3)-N-acetylgalactosaminyltransferase (GTA) and α-(1 → 3)-galactosyltransferase (GTB) mutants, by mass spectrometry (MS) is reported. Incubation of mutants of GTA or GTB, in which the putative catalytic nucleophile Glu(303) was replaced with Cys (i.e. GTA(E303C) and GTB(E303C)), with their respective donor substrate results in a covalent intermediate. Tandem MS analysis using collision-induced dissociation confirmed Cys(303) as the site of glycosylation. Exposure of the glycosyl-enzyme intermediates to a disaccharide acceptor results in the formation of the corresponding enzymatic trisaccharide products. These findings suggest that the GTA(E303C) and GTB(E303C) mutants may operate by a double-displacement mechanism.
authors.affiliation: Alberta Ingenuity Centre for Carbohydrate Science, Department of Chemistry, University of Alberta, Edmonton, Alta, Canada.
authors.forename: Naoto
authors.initials: N
authors.lastname: Soya
authors.forename: Ying
authors.initials: Y
authors.lastname: Fang
authors.forename: Monica M
authors.initials: MM
authors.lastname: Palcic
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization; D014312:Trisaccharides / Q000378:metabolism
chemical_list: D009005:Monosaccharides; D014312:Trisaccharides; D005700:Galactosyltransferases