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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.

Original publication

DOI

10.1038/nchembio.628

Type

Journal article

Journal

Nat Chem Biol

Publication Date

07/08/2011

Volume

7

Pages

631 - 638

Keywords

Catalysis, Catalytic Domain, Glucosyltransferases, Hydrogen Bonding, Models, Molecular