title: Mechanistic analysis of trehalose synthase from Mycobacterium smegmatis.
abstract: Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose and has been shown recently to function primarily in the mobilization of trehalose as a glycogen precursor. Consequently, the mechanism of this intriguing isomerase is of both academic and potential pharmacological interest. TreS catalyzes the hydrolytic cleavage of α-aryl glucosides as well as α-glucosyl fluoride, thereby allowing facile, continuous assays. Reaction of TreS with 5-fluoroglycosyl fluorides results in the trapping of a covalent glycosyl-enzyme intermediate consistent with TreS being a member of the retaining glycoside hydrolase family 13 enzyme family, thus likely following a two-step, double displacement mechanism. This trapped intermediate was subjected to protease digestion followed by LC-MS/MS analysis, and Asp(230) was thereby identified as the catalytic nucleophile. The isomerization reaction was shown to be an intramolecular process by demonstration of the inability of TreS to incorporate isotope-labeled exogenous glucose into maltose or trehalose consistent with previous studies on other TreS enzymes. The absence of a secondary deuterium kinetic isotope effect and the general independence of k(cat) upon leaving group ability both point to a rate-determining conformational change, likely the opening and closing of the enzyme active site.
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Ran
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Yuan T
authors.initials: YT
authors.lastname: Pan
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Shouming
authors.initials: S
authors.lastname: He
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Lam
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Brayer
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Elbein
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada. Electronic address: withers@chem.ubc.ca.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005964:Glucosyltransferases; C099692:trehalose synthase; D006026:Glycoside Hydrolases