Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase
Seung Seo Lee1, Sung You Hong, James C Errey
1Department of Chemistry, University of Oxford, Oxford, United Kingdom.
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.
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