title: Mechanistic studies of a retaining alpha-galactosyltransferase from Neisseria meningitidis.
abstract: Lipopolysaccharyl alpha-galactosyltransferase from Neisseria meningitidis catalyzes the transfer of a galactosyl moiety from the activated donor UDP-Gal to glycoconjugates to yield an elongated saccharide product with net retention of anomeric configuration relative to the donor substrate. Through kinetic analyses in which the concentrations of both substrates are independently varied and through inhibition studies with dead-end analogues of both substrates and with the oligosaccharide product, we have demonstrated that this enzyme follows an ordered bi-bi kinetic mechanism. Various aspects of the chemical mechanism including the possible formation of a covalent glycosyl-enzyme intermediate were also probed using an assortment of strategies. While the results of these investigations were unable to clearly delineate the chemical mechanism of this enzyme, they provide important insights into the catalytic machinery surrounding the events involved in catalysis.
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1 Canada.
authors.forename: Hoa D
authors.initials: HD
authors.lastname: Ly
authors.forename: Brenda
authors.initials: B
authors.lastname: Lougheed
authors.forename: Warren W
authors.initials: WW
authors.lastname: Wakarchuk
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D016695:Glycosyltransferases / Q000737:chemistry; D007700:Kinetics; D009345:Neisseria meningitidis / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010103:Oxygen Isotopes / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014531:Uridine Diphosphate Galactose / Q000737:chemistry; D000519:alpha-Galactosidase / Q000737:chemistry*
chemical_list: D010103:Oxygen Isotopes; D011994:Recombinant Proteins; D014531:Uridine Diphosphate Galactose; D016695:Glycosyltransferases; D000519:alpha-Galactosidase