Mechanistic evidence for a front-side, S(N)i-type reaction in a retaining glycosyltransferase

Seung Seo Lee; Sung You Hong; James C. Errey; Atsushi Izumi; Gideon J. Davies; Benjamin G. Davis

doi:10.1038/NCHEMBIO.628

Mechanistic evidence for a front-side, S(N)i-type reaction in a retaining glycosyltransferase

Seung Seo Lee^*, Sung You Hong, James C. Errey, Atsushi Izumi, Gideon J. Davies, Benjamin G. Davis

^*Corresponding author for this work

Chemistry

Research output: Contribution to journal › Article › peer-review

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 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.

Original language	English
Pages (from-to)	631-638
Number of pages	8
Journal	NATURE CHEMICAL BIOLOGY
Volume	7
Issue number	9
DOIs	https://doi.org/10.1038/NCHEMBIO.628
Publication status	Published - Sept 2011

Keywords

COVALENT INTERMEDIATE
SUBSTRATE-ASSISTED CATALYSIS
ALPHA-GALACTOSYLTRANSFERASE
ENZYMATIC GLYCOSYL TRANSFER
SCHIZOPHYLLUM-COMMUNE
NEISSERIA-MENINGITIDIS
GLUCOSYL TRANSFER
TRANSITION-STATE
TREHALOSE PHOSPHORYLASE
ACTIVE-SITE

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10.1038/NCHEMBIO.628

Cite this

@article{1cf8be80d4734b449b37d4062d41c744,

title = "Mechanistic evidence for a front-side, S(N)i-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 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.",

keywords = "COVALENT INTERMEDIATE, SUBSTRATE-ASSISTED CATALYSIS, ALPHA-GALACTOSYLTRANSFERASE, ENZYMATIC GLYCOSYL TRANSFER, SCHIZOPHYLLUM-COMMUNE, NEISSERIA-MENINGITIDIS, GLUCOSYL TRANSFER, TRANSITION-STATE, TREHALOSE PHOSPHORYLASE, ACTIVE-SITE",

author = "Lee, {Seung Seo} and Hong, {Sung You} and Errey, {James C.} and Atsushi Izumi and Davies, {Gideon J.} and Davis, {Benjamin G.}",

year = "2011",

month = sep,

doi = "10.1038/NCHEMBIO.628",

language = "English",

volume = "7",

pages = "631--638",

journal = "NATURE CHEMICAL BIOLOGY",

issn = "1552-4450",

publisher = "Nature Publishing Group",

number = "9",

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AU - Lee, Seung Seo

AU - Hong, Sung You

AU - Errey, James C.

AU - Izumi, Atsushi

AU - Davies, Gideon J.

AU - Davis, Benjamin G.

PY - 2011/9

Y1 - 2011/9

N2 - 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.

AB - 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.

KW - COVALENT INTERMEDIATE

KW - SUBSTRATE-ASSISTED CATALYSIS

KW - ALPHA-GALACTOSYLTRANSFERASE

KW - ENZYMATIC GLYCOSYL TRANSFER

KW - SCHIZOPHYLLUM-COMMUNE

KW - NEISSERIA-MENINGITIDIS

KW - GLUCOSYL TRANSFER

KW - TRANSITION-STATE

KW - TREHALOSE PHOSPHORYLASE

KW - ACTIVE-SITE

U2 - 10.1038/NCHEMBIO.628

DO - 10.1038/NCHEMBIO.628

M3 - Article

SN - 1552-4450

VL - 7

SP - 631

EP - 638

JO - NATURE CHEMICAL BIOLOGY

JF - NATURE CHEMICAL BIOLOGY

IS - 9

ER -