title: Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase.
abstract: A previously determined crystal structure of the ternary complex of trehalose-6-phosphate synthase identified a putative transition state-like arrangement based on validoxylamine A 6'-O-phosphate and uridine diphosphate in the active site. Here linear free energy relationships confirm that these inhibitors are synergistic transition state mimics, supporting front-face nucleophilic attack involving hydrogen bonding between leaving group and nucleophile. Kinetic isotope effects indicate a highly dissociative oxocarbenium ion-like transition state. Leaving group (18)O effects identified isotopically sensitive bond cleavages and support the existence of a hydrogen bond between the nucleophile and departing group. Brønsted analysis of nucleophiles and Taft analysis highlight participation of the nucleophile in the transition state, also consistent with a front-face mechanism. Together, these comprehensive, quantitative data substantiate this unusual enzymatic reaction mechanism. Its discovery should prompt useful reassessment of many biocatalysts and their substrates and inhibitors.
authors.affiliation: Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Seung Seo
authors.initials: SS
authors.lastname: Lee
authors.forename: Sung You
authors.initials: SY
authors.lastname: Hong
authors.forename: James C
authors.initials: JC
authors.lastname: Errey
authors.forename: Atsushi
authors.initials: A
authors.lastname: Izumi
authors.forename: Gideon J
authors.initials: GJ
authors.lastname: Davies
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Davis
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D005964:Glucosyltransferases / Q000737:chemistry*; D006860:Hydrogen Bonding; D008958:Models, Molecular
chemical_list: D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase