Release of halide ions from the buried active site of the haloalkane dehalogenase LinB revealed by stopped-flow fluorescence analysis and free energy calculations
| Autor: | Jiri Damborsky, Jana Hladílková, Zbynek Prokop, Pavel Jungwirth, Radka Chaloupková |
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| Rok vydání: | 2013 |
| Předmět: |
Haloalkane
Hydrolases Halide Protonation Molecular Dynamics Simulation 010402 general chemistry Photochemistry 01 natural sciences 03 medical and health sciences Deprotonation Halogens Catalytic Domain Materials Chemistry Physical and Theoretical Chemistry Anion binding 030304 developmental biology chemistry.chemical_classification Ions 0303 health sciences biology Active site 0104 chemical sciences Surfaces Coatings and Films Kinetics Spectrometry Fluorescence chemistry Catalytic cycle biology.protein Thermodynamics Haloalkane dehalogenase Protein Binding |
| Zdroj: | The journal of physical chemistry. B. 117(46) |
| ISSN: | 1520-5207 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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