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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F12%3A00057142" target="_blank" >RIV/00216224:14310/12:00057142 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1074/jbc.M112.377853" target="_blank" >http://dx.doi.org/10.1074/jbc.M112.377853</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1074/jbc.M112.377853" target="_blank" >10.1074/jbc.M112.377853</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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 ischanged 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-limitingstep of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally.

Název v anglickém jazyce

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

Popis výsledku anglicky

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 ischanged 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-limitingstep of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally.

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

EB - Genetika a molekulární biologie

OECD FORD obor

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Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2012

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

The Journal of Biological Chemistry

ISSN

0021-9258

e-ISSN

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Svazek periodika

287

Číslo periodika v rámci svazku

34

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

13

Strana od-do

29062-29074

Kód UT WoS článku

000308074600073

EID výsledku v databázi Scopus

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