Mechanistic evidence for a front-side, S(N)i-type reaction in a retaining glycosyltransferase
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- Mechanistic evidence for a front-side, S(N)i-type reaction in a retaining glycosyltransferase
- Lee, Seung Seo; Hong, Sung You; Errey, James C.; Izumi, Atsushi; Davies, Gideon J.; Davis, Benjamin G.
- SUBSTRATE-ASSISTED CATALYSIS; ENZYMATIC GLYCOSYL TRANSFER; TRANSITION-STATE; ALPHA-GALACTOSYLTRANSFERASE; TREHALOSE PHOSPHORYLASE; NEISSERIA-MENINGITIDIS; SCHIZOPHYLLUM-COMMUNE; COVALENT INTERMEDIATE; GLUCOSYL TRANSFER; ACTIVE-SITE
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- NATURE PUBLISHING GROUP
- NATURE CHEMICAL BIOLOGY, v.7, no.9, pp.631 - 638
- 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 O-18 effects identified isotopically sensitive bond cleavages and support the existence of a hydrogen bond between the nucleophile and departing group. Bronsted 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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