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Title: Mutational, Structural, and Kinetic Evidence for a Dissociative Mechanism in the GDP-mannose Mannosyl Hydrolase Reaction

Abstract

GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-{alpha}-D-sugars by nucleophilic substitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124. Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.3 Angstrom X-ray structure of the GDPMH-Mg{sup 2+}-GDP{center_dot}Tris{sup +} complex, the GDP leaving group interacts with five catalytic components: R37, Y103, R52, R65, and the essential Mg{sup 2+}. As determined by the effects of site-specific mutants on k{sub cat}, these components contribute factors of 24-, 100-, 309-, 24-, and {ge}10{sup 5}-fold, respectively, to catalysis. Both R37 and Y103 bind the {beta}-phosphate of GDP and are only 5.0 Angstroms apart. Accordingly, the R37Q/Y103F double mutant exhibits partially additive effects of the two single mutants on k{sub cat}, indicating cooperativity of R37 and Y103 in promoting catalysis, and antagonistic effects on K{sub m}. Second, the conserved residue, D22, is positioned to accept a hydrogen bond from the C2-OH group of the sugar undergoing substitution at C1, as was shown by modeling an {alpha}-D-mannosyl group into the sugar binding site. The D22A and D22N mutations decreased k{sub cat} by factors of 10{sup 2.1} and 10{sup 2.6}, respectively, for the hydrolysis of GDP-{alpha}-D-mannose, andmore » showed smaller effects on K{sub m}, suggesting that the D22 anion stabilizes a cationic oxocarbenium transition state. Third, the fluorinated substrate, GDP-2F-{alpha}-D-mannose, for which a cationic oxocarbenium transition state would be destabilized by electron withdrawal, exhibited a 16-fold decrease in k{sub cat} and a smaller, 2.5-fold increase in K{sub m}. The D22A and D22N mutations further decreased the k{sub cat} with GDP-2F-{alpha}-D-mannose to values similar to those found with GDP-{alpha}-D-mannose, and decreased the K{sub m} of the fluorinated substrate. The choice of histidine as the general base over glutamate, the preferred base in other Nudix enzymes, is not due to the greater basicity of histidine, since the pK{sub a} of E124 in the active complex (7.7) exceeded that of H124 (6.7), and the H124E mutation showed a 10{sup 2.2}-fold decrease in k{sub cat}and a 4.0-fold increase in K{sub m} at pH 9.3. Similarly, the catalytic triad detected in the X-ray structure (H124---Y127---P120) is unnecessary for orienting H124, since the Y127F mutation had only 2-fold effects on k{sub cat} and K{sub m} with either H124 or E124 as the general base. Hence, a neutral histidine rather than an anionic glutamate may be necessary to preserve electroneutrality in the active complex.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
913718
Report Number(s):
BNL-78286-2007-JA
Journal ID: ISSN 0006-2960; BICHAW; TRN: US200804%%172
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 44; Journal Issue: 25; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
08 HYDROGEN; ADDITIVES; ANIONS; ATOMS; CATALYSIS; ELECTRONS; ENZYMES; HISTIDINE; HYDROGEN; HYDROLASES; HYDROLYSIS; KINETICS; MUTANTS; MUTATIONS; SACCHAROSE; national synchrotron light source

Citation Formats

Xia, Z, Azurmendi, H, lairson, L, Withers, S, Gabelli, S, Bianchet, M, Amzel, L, and Mildvan, A. Mutational, Structural, and Kinetic Evidence for a Dissociative Mechanism in the GDP-mannose Mannosyl Hydrolase Reaction. United States: N. p., 2005. Web. doi:10.1021/bi050583v.
Xia, Z, Azurmendi, H, lairson, L, Withers, S, Gabelli, S, Bianchet, M, Amzel, L, & Mildvan, A. Mutational, Structural, and Kinetic Evidence for a Dissociative Mechanism in the GDP-mannose Mannosyl Hydrolase Reaction. United States. https://doi.org/10.1021/bi050583v
Xia, Z, Azurmendi, H, lairson, L, Withers, S, Gabelli, S, Bianchet, M, Amzel, L, and Mildvan, A. Sat . "Mutational, Structural, and Kinetic Evidence for a Dissociative Mechanism in the GDP-mannose Mannosyl Hydrolase Reaction". United States. https://doi.org/10.1021/bi050583v.
@article{osti_913718,
title = {Mutational, Structural, and Kinetic Evidence for a Dissociative Mechanism in the GDP-mannose Mannosyl Hydrolase Reaction},
author = {Xia, Z and Azurmendi, H and lairson, L and Withers, S and Gabelli, S and Bianchet, M and Amzel, L and Mildvan, A},
abstractNote = {GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-{alpha}-D-sugars by nucleophilic substitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124. Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.3 Angstrom X-ray structure of the GDPMH-Mg{sup 2+}-GDP{center_dot}Tris{sup +} complex, the GDP leaving group interacts with five catalytic components: R37, Y103, R52, R65, and the essential Mg{sup 2+}. As determined by the effects of site-specific mutants on k{sub cat}, these components contribute factors of 24-, 100-, 309-, 24-, and {ge}10{sup 5}-fold, respectively, to catalysis. Both R37 and Y103 bind the {beta}-phosphate of GDP and are only 5.0 Angstroms apart. Accordingly, the R37Q/Y103F double mutant exhibits partially additive effects of the two single mutants on k{sub cat}, indicating cooperativity of R37 and Y103 in promoting catalysis, and antagonistic effects on K{sub m}. Second, the conserved residue, D22, is positioned to accept a hydrogen bond from the C2-OH group of the sugar undergoing substitution at C1, as was shown by modeling an {alpha}-D-mannosyl group into the sugar binding site. The D22A and D22N mutations decreased k{sub cat} by factors of 10{sup 2.1} and 10{sup 2.6}, respectively, for the hydrolysis of GDP-{alpha}-D-mannose, and showed smaller effects on K{sub m}, suggesting that the D22 anion stabilizes a cationic oxocarbenium transition state. Third, the fluorinated substrate, GDP-2F-{alpha}-D-mannose, for which a cationic oxocarbenium transition state would be destabilized by electron withdrawal, exhibited a 16-fold decrease in k{sub cat} and a smaller, 2.5-fold increase in K{sub m}. The D22A and D22N mutations further decreased the k{sub cat} with GDP-2F-{alpha}-D-mannose to values similar to those found with GDP-{alpha}-D-mannose, and decreased the K{sub m} of the fluorinated substrate. The choice of histidine as the general base over glutamate, the preferred base in other Nudix enzymes, is not due to the greater basicity of histidine, since the pK{sub a} of E124 in the active complex (7.7) exceeded that of H124 (6.7), and the H124E mutation showed a 10{sup 2.2}-fold decrease in k{sub cat}and a 4.0-fold increase in K{sub m} at pH 9.3. Similarly, the catalytic triad detected in the X-ray structure (H124---Y127---P120) is unnecessary for orienting H124, since the Y127F mutation had only 2-fold effects on k{sub cat} and K{sub m} with either H124 or E124 as the general base. Hence, a neutral histidine rather than an anionic glutamate may be necessary to preserve electroneutrality in the active complex.},
doi = {10.1021/bi050583v},
url = {https://www.osti.gov/biblio/913718}, journal = {Biochemistry},
issn = {0006-2960},
number = 25,
volume = 44,
place = {United States},
year = {2005},
month = {1}
}