title: Mechanistic differences among retaining disaccharide phosphorylases: insights from kinetic analysis of active site mutants of sucrose phosphorylase and alpha,alpha-trehalose phosphorylase.
abstract: Sucrose phosphorylase utilizes a glycoside hydrolase-like double displacement mechanism to convert its disaccharide substrate and phosphate into alpha-d-glucose 1-phosphate and fructose. Site-directed mutagenesis was employed to characterize the proposed roles of Asp(196) and Glu(237) as catalytic nucleophile and acid-base, respectively, in the reaction of sucrose phosphorylase from Leuconostoc mesenteroides. The side chain of Asp(295) is suggested to facilitate the catalytic steps of glucosylation and deglucosylation of Asp(196) through a strong hydrogen bond (23 kJ/mol) with the 2-hydroxyl of the glucosyl oxocarbenium ion-like species believed to be formed in the transition states flanking the beta-glucosyl enzyme intermediate. An assortment of biochemical techniques used to examine the mechanism of alpha-retaining glucosyl transfer by Schizophyllum commune alpha,alpha-trehalose phosphorylase failed to provide evidence in support of a similar two-step catalytic reaction via a covalent intermediate. Mutagenesis studies suggested a putative active-site structure for this trehalose phosphorylase that is typical of retaining glycosyltransferases of fold family GT-B and markedly different from that of sucrose phosphorylase. While ambiguity remains regarding the chemical mechanism by which the trehalose phosphorylase functions, the two disaccharide phosphorylases have evolved strikingly different reaction coordinates to achieve catalytic efficiency and stereochemical control in their highly analogous substrate transformations.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
authors.forename: Christiane
authors.initials: C
authors.lastname: Goedl
authors.forename: Alexandra
authors.initials: A
authors.lastname: Schwarz
authors.forename: Mario
authors.initials: M
authors.lastname: Mueller
authors.forename: Lothar
authors.initials: L
authors.lastname: Brecker
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D000363:Agaricales / Q000201:enzymology; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D005632:Fructose / Q000378:metabolism; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
chemical_list: D010710:Phosphates; D005632:Fructose; D001224:Aspartic Acid; D018698:Glutamic Acid; D014638:Vanadates; D005964:Glucosyltransferases; C023181:alpha,alpha-trehalose phosphorylase; C025901:sucrose phosphorylase