A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB

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Publikace nespadá pod Ekonomicko-správní fakultu, ale pod Přírodovědeckou fakultu. Oficiální stránka publikace je na webu muni.cz.
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BIEDERMANNOVÁ Lada PROKOP Zbyněk GORA Artur Wiktor CHOVANCOVÁ Eva KOVÁCS Mihály DAMBORSKÝ Jiří WADE Rebecca C.

Rok publikování 2012
Druh Článek v odborném periodiku
Časopis / Zdroj The Journal of Biological Chemistry
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
Doi http://dx.doi.org/10.1074/jbc.M112.377853
Obor Genetika a molekulární biologie
Klíčová slova 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
Popis 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.
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