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Release of Halide Ions from the Buried Active Site of the Haloalkane Dehalogenase LinB Revealed by Stopped-Flow Fluorescence Analysis and Free Energy Calculations
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SYSNO ASEP 0421950 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Release of Halide Ions from the Buried Active Site of the Haloalkane Dehalogenase LinB Revealed by Stopped-Flow Fluorescence Analysis and Free Energy Calculations Author(s) Hladílková, Jana (UOCHB-X) RID
Prokop, Z. (CZ)
Chaloupková, R. (CZ)
Damborský, J. (CZ)
Jungwirth, Pavel (UOCHB-X) RID, ORCIDNumber of authors 5 Source Title Journal of Physical Chemistry B. - : American Chemical Society - ISSN 1520-6106
Roč. 117, č. 46 (2013), s. 14329-14335Number of pages 7 s. Language eng - English Country US - United States Keywords access tunnel ; buried active site ; catalytic activity ; enzyme mechanism ; haloalkane dehalogenase ; halide ions Subject RIV CF - Physical ; Theoretical Chemistry R&D Projects GBP208/12/G016 GA ČR - Czech Science Foundation (CSF) Institutional support UOCHB-X - RVO:61388963 UT WOS 000327557700015 EID SCOPUS 84888618153 DOI 10.1021/jp409040u Annotation Release of halide ions is an essential step of the catalytic cycle of haloalkane dehalogenases. Here we describe experimentally and computationally the process of release of a halide anion from the buried active site of the haloalkane dehalogenase LinB. Using stopped-flow fluorescence analysis and umbrella sampling free energy calculations, we show that the anion binding is ion-specific and follows the ordering I-_(-)> Br- > Cl- .We also address the issue of the protonation state of the catalytic His272 residue and its effect on the process of halide release. While deprotonation of His272 increases binding of anions in the access tunnel, we show that the anionic ordering does not change with the switch of the protonation state. We also demonstrate that a sodium cation could relatively easily enter the active site, provided the His272 residue is singly protonated, and replace thus the missing proton. In contrast, Na+ is strongly repelled from the active site containing the doubly protonated His272 residue. Our study contributes toward understanding of the reaction mechanism of haloalkane dehalogenase enzyme family. Determination of the protonation state of the catalytic histidine throughout the catalytic cycle remains a challenge for future studies. Workplace Institute of Organic Chemistry and Biochemistry Contact asep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Viktorie Chládková, Tel.: 232 002 434 Year of Publishing 2014
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