A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB

• Verfasst von: Biedermannová, Lada [VerfasserIn]
• Verfasst von: Wade, Rebecca C. [VerfasserIn]
• Titel: A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB
• Verf.angabe: Lada Biedermannová, Zbyněk Prokop, Artur Gora, Eva Chovancová, Mihály Kovács, Jiří Damborský and Rebecca C. Wade
• E-Jahr: 2012
• Jahr: June 28, 2012
• Umfang: 13 S.
• Fussnoten: First published on June 28, 2012 ; Gesehen am 03.07.2018
• Titel Quelle: Enthalten in: The journal of biological chemistry
• Ort Quelle: Bethesda, Md. : Soc., 1905
• Jahr Quelle: 2012
• Band/Heft Quelle: 287(2012), 34, Seite 29062-29074
• ISSN Quelle: 1083-351X
• DOI: doi:10.1074/jbc.M112.377853
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: Bromide Ion
• Sach-SW: Computer Modeling
• Sach-SW: Enzyme Kinetics
• Sach-SW: Enzyme Mechanisms
• Sach-SW: Free Energy Profile
• Sach-SW: Haloalkane Dehalogenase
• Sach-SW: Molecular Dynamics
• Sach-SW: Product Release
• Sach-SW: Protein Engineering
• Sach-SW: Transient Kinetics
• K10plus-PPN: 157723801X

Abstract

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.