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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

Hladilkova, Jana LU ; Prokop, Zbynek ; Chaloupkova, Radka ; Damborsky, Jiri and Jungwirth, Pavel (2013) In Journal of Physical Chemistry B 117(46). p.14329-14335
Abstract

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... (More)

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.

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author
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publishing date
type
Contribution to journal
publication status
published
in
Journal of Physical Chemistry B
volume
117
issue
46
pages
7 pages
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:84888618153
  • pmid:24151979
ISSN
1520-6106
DOI
10.1021/jp409040u
language
English
LU publication?
no
id
c35001ad-0c68-46ea-9e1d-2d05d6b4381d
date added to LUP
2025-07-18 11:20:40
date last changed
2025-08-28 10:34:18
@article{c35001ad-0c68-46ea-9e1d-2d05d6b4381d,
  abstract     = {{<p>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<sup>-</sup> &gt; Br<sup>-</sup> &gt; Cl<sup>-</sup>. 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<sup>+</sup> 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.</p>}},
  author       = {{Hladilkova, Jana and Prokop, Zbynek and Chaloupkova, Radka and Damborsky, Jiri and Jungwirth, Pavel}},
  issn         = {{1520-6106}},
  language     = {{eng}},
  month        = {{11}},
  number       = {{46}},
  pages        = {{14329--14335}},
  publisher    = {{The American Chemical Society (ACS)}},
  series       = {{Journal of Physical Chemistry B}},
  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}},
  url          = {{http://dx.doi.org/10.1021/jp409040u}},
  doi          = {{10.1021/jp409040u}},
  volume       = {{117}},
  year         = {{2013}},
}