| BIEDERMANNOVÁ, Lada, Zbyněk PROKOP, Artur Wiktor GORA, Eva CHOVANCOVÁ, Mihály KOVÁCS, Jiří DAMBORSKÝ and Rebecca C. WADE. A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB. The Journal of Biological Chemistry. USA, 2012, vol. 287, No 34, p. 29062-29074. ISSN 0021-9258. Available from: https://dx.doi.org/10.1074/jbc.M112.377853. |
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@article{1082672,
author = {Biedermannová, Lada and Prokop, Zbyněk and Gora, Artur Wiktor and Chovancová, Eva and Kovács, Mihály and Damborský, Jiří and Wade, Rebecca C.},
article_location = {USA},
article_number = {34},
doi = {http://dx.doi.org/10.1074/jbc.M112.377853},
keywords = {HLD; haloalkane dehalogenase; MD; molecular dynamics; RAMD; random acceleration molecular dynamics; ABF; adaptive biasing force; RC; reaction coordinate; FEP; free energy perturbation; NATA; N-acetyltryptophan amide},
language = {eng},
issn = {0021-9258},
journal = {The Journal of Biological Chemistry},
title = {A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB},
volume = {287},
year = {2012}
}
TY - JOUR
ID - 1082672
AU - Biedermannová, Lada - Prokop, Zbyněk - Gora, Artur Wiktor - Chovancová, Eva - Kovács, Mihály - Damborský, Jiří - Wade, Rebecca C.
PY - 2012
TI - A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB
JF - The Journal of Biological Chemistry
VL - 287
IS - 34
SP - 29062-29074
EP - 29062-29074
SN - 00219258
KW - HLD
KW - haloalkane dehalogenase
KW - MD
KW - molecular dynamics
KW - RAMD
KW - random acceleration molecular dynamics
KW - ABF
KW - adaptive biasing force
KW - RC
KW - reaction coordinate
KW - FEP
KW - free energy perturbation
KW - NATA
KW - N-acetyltryptophan amide
N2 - Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.
ER -
BIEDERMANNOVÁ, Lada, Zbyněk PROKOP, Artur Wiktor GORA, Eva CHOVANCOVÁ, Mihály KOVÁCS, Jiří DAMBORSKÝ and Rebecca C. WADE. A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB. \textit{The Journal of Biological Chemistry}. USA, 2012, vol.~287, No~34, p.~29062-29074. ISSN~0021-9258. Available from: https://dx.doi.org/10.1074/jbc.M112.377853.
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