Kinetics of binding of fluorescent ligands to enzymes with engineered access tunnels

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00159816%3A_____%2F17%3A00066800" target="_blank" >RIV/00159816:_____/17:00066800 - isvavai.cz</a>

Alternative codes found

RIV/00216224:14310/17:00095411

Result on the web

<a href="http://dx.doi.org/10.1111/febs.13957" target="_blank" >http://dx.doi.org/10.1111/febs.13957</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1111/febs.13957" target="_blank" >10.1111/febs.13957</a>

Result language

angličtina

Original language name

Kinetics of binding of fluorescent ligands to enzymes with engineered access tunnels

Original language description

Molecular recognition mechanisms and kinetics of binding of ligands to buried active sites via access tunnels are not well understood. Fluorescence polarization enables rapid and non-destructive real-time quantification of the association between small fluorescent ligands and large biomolecules. In this study, we describe analysis of binding kinetics of fluorescent ligands resembling linear halogenated alkanes to haloalkane dehalogenases. Dehalogenases possess buried active sites connected to the surrounding solvent by access tunnels. Modification of these tunnels by mutagenesis has emerged as a novel strategy to tailor the enzyme properties. We demonstrate that the fluorescence polarization method can sense differences in binding kinetics originating from even single mutations introduced to the tunnels. The results show, strikingly, that the rate constant of the dehalogenase variants varied across seven orders of magnitude, and the type of ligand used strongly affected the binding kinetics of the enzyme. Furthermore, fluorescence polarization could be applied to cell-free extracts instead of purified proteins, extending the method&apos;s application to medium-throughput screening of enzyme variant libraries generated in directed evolution experiments. The method can also provide in-depth kinetic information about the rate-determining step in binding kinetics and reveals the bottlenecks of enzyme accessibility. Assuming availability of appropriate fluorescent ligand, the method could be applied for analysis of accessibility of tunnels and buried active sites of enzymes forming a covalent alkyl-enzyme intermediate during their catalytic cycle, such as α/β-hydrolases containing &gt; 100 000 protein sequences based on the Pfam database.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

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OECD FORD branch

10608 - Biochemistry and molecular biology

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

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

Publication year

2017

Confidentiality

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

Name of the periodical

FEBS Journal

ISSN

1742-464X

e-ISSN

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Volume of the periodical

284

Issue of the periodical within the volume

1

Country of publishing house

US - UNITED STATES

Number of pages

15

Pages from-to

134-148

UT code for WoS article

000393601200010

EID of the result in the Scopus database

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