title: A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB.
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.
authors: Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies, Schloss-Wolfsbrunnenweg 35, D-69118 Heidelberg, Germany. lada.biedermannova@img.cas.cz
authors: Lada
authors: L
authors: Biedermannová
authors: Zbyněk
authors: Z
authors: Prokop
authors: Artur
authors: A
authors: Gora
authors: Eva
authors: E
authors: Chovancová
authors: Mihály
authors: M
authors: Kovács
authors: Jiří
authors: J
authors: Damborsky
authors: Rebecca C
authors: RC
authors: Wade
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D042301:Sphingomonadaceae / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D006867:Hydrolases; C046137:haloalkane dehalogenase