Molecular Gating of an Engineered Enzyme Captured in Real Time

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F18%3A00101750" target="_blank" >RIV/00216224:14310/18:00101750 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/61388971:_____/18:00500166 RIV/61388955:_____/18:00498928 RIV/00159816:_____/18:00069367

Výsledek na webu

<a href="http://dx.doi.org/10.1021/jacs.8b09848" target="_blank" >http://dx.doi.org/10.1021/jacs.8b09848</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/jacs.8b09848" target="_blank" >10.1021/jacs.8b09848</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Molecular Gating of an Engineered Enzyme Captured in Real Time

Popis výsledku v původním jazyce

Enzyme engineering tends to focus on the design of active sites for the chemical steps, while the physical steps of the catalytic cycle are often overlooked. Tight binding of a substrate in an active site is beneficial for the chemical steps, whereas good accessibility benefits substrate binding and product release. Many enzymes control the accessibility of their active sites by molecular gates. Here we analyzed the dynamics of a molecular gate artificially introduced into an access tunnel of the most efficient haloalkane dehalogenase using pre-steady-state kinetics, single-molecule fluorescence spectroscopy, and molecular dynamics. Photoinduced electron-transfer fluorescence correlation spectroscopy (PET-FCS) has enabled real-time observation of molecular gating at the single-molecule level with rate constants (k(on) = 1822 s(-1), k(off) = 60 s(-1)) corresponding well with those from the pre-steady-state kinetics (k(-1) = 1100 s(-1), k(1) = 20 s(-1)). The PET-FCS technique is used here to study the conformational dynamics in a soluble enzyme, thus demonstrating an additional application for this method. Engineering dynamical molecular gates represents a widely applicable strategy for designing efficient biocatalysts.

Název v anglickém jazyce

Molecular Gating of an Engineered Enzyme Captured in Real Time

Popis výsledku anglicky

Enzyme engineering tends to focus on the design of active sites for the chemical steps, while the physical steps of the catalytic cycle are often overlooked. Tight binding of a substrate in an active site is beneficial for the chemical steps, whereas good accessibility benefits substrate binding and product release. Many enzymes control the accessibility of their active sites by molecular gates. Here we analyzed the dynamics of a molecular gate artificially introduced into an access tunnel of the most efficient haloalkane dehalogenase using pre-steady-state kinetics, single-molecule fluorescence spectroscopy, and molecular dynamics. Photoinduced electron-transfer fluorescence correlation spectroscopy (PET-FCS) has enabled real-time observation of molecular gating at the single-molecule level with rate constants (k(on) = 1822 s(-1), k(off) = 60 s(-1)) corresponding well with those from the pre-steady-state kinetics (k(-1) = 1100 s(-1), k(1) = 20 s(-1)). The PET-FCS technique is used here to study the conformational dynamics in a soluble enzyme, thus demonstrating an additional application for this method. Engineering dynamical molecular gates represents a widely applicable strategy for designing efficient biocatalysts.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10401 - Organic chemistry

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

2018

Kód důvěrnosti údajů

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

Název periodika

Journal of the American Chemical Society

ISSN

0002-7863

e-ISSN

—

Svazek periodika

140

Číslo periodika v rámci svazku

51

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

10

Strana od-do

17999-18008

Kód UT WoS článku

000454751800028

EID výsledku v databázi Scopus

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