J 2012

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

BIEDERMANNOVÁ, Lada, Zbyněk PROKOP, Artur Wiktor GORA, Eva CHOVANCOVÁ, Mihály KOVÁCS et. al.

Basic information

Original name

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

Authors

BIEDERMANNOVÁ, Lada (203 Czech Republic), Zbyněk PROKOP (203 Czech Republic, belonging to the institution), Artur Wiktor GORA (616 Poland, belonging to the institution), Eva CHOVANCOVÁ (203 Czech Republic, belonging to the institution), Mihály KOVÁCS (348 Hungary), Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution) and Rebecca C. WADE (276 Germany)

Edition

The Journal of Biological Chemistry, USA, 2012, 0021-9258

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

Genetics and molecular biology

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

Impact factor

Impact factor: 4.651

RIV identification code

RIV/00216224:14310/12:00057142

Organization unit

Faculty of Science

UT WoS

000308074600073

Keywords in English

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

Tags

Změněno: 22/4/2013 15:09, Ing. Andrea Mikešková

Abstract

V originále

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.

Links

ED0001/01/01, research and development project
Name: CETOCOEN
IAA401630901, research and development project
Name: Evoluce substrátové specifity u enzymů aktivních s xenobiotickými látkami
Investor: Academy of Sciences of the Czech Republic, Evolution of substrate specificity in enzymes acting on xenobiotic compounds