Document Detail


A substrate-assisted mechanism of nucleophile activation in a Ser-His-Asp containing C-C bond hydrolase.
MedLine Citation:
PMID:  24067021     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The meta-cleavage product (MCP) hydrolases utilize a Ser-His-Asp triad to hydrolyze a carbon-carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES(red), which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES(red) decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2 (βnuc ∼ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His-Asp pair does not play an essential role. The data further suggest that ES(red) represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.
Authors:
Antonio C Ruzzini; Shiva Bhowmik; Subhangi Ghosh; Katherine C Yam; Jeffrey T Bolin; Lindsay D Eltis
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2013-10-09
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Oct 
Date Detail:
Created Date:  2013-10-22     Completed Date:  2013-12-12     Revised Date:  2014-06-03    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  7428-38     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Acylation
Aspartic Acid / chemistry*,  metabolism
Bacterial Proteins / chemistry*,  metabolism
Catalysis
Chromatography, Liquid
Dipeptides / chemistry*,  metabolism
Fatty Acids, Unsaturated / chemistry*,  metabolism
Hydrolases / chemistry*,  metabolism
Hydrolysis
Kinetics
Models, Chemical
Spectrometry, Mass, Electrospray Ionization
Sphingomonas / enzymology*
Substrate Specificity
Grant Support
ID/Acronym/Agency:
P41 GM103543/GM/NIGMS NIH HHS; P41GM103543/GM/NIGMS NIH HHS; R24 GM111072/GM/NIGMS NIH HHS; RR007707/RR/NCRR NIH HHS
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/Dipeptides; 0/Fatty Acids, Unsaturated; 0/seryl-histidine; 30KYC7MIAI/Aspartic Acid; 50480-67-6/2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid; EC 3.-/Hydrolases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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