J 2018

Molecular Gating of an Engineered Enzyme Captured in Real Time

KOKKONEN, Piia Pauliina, J. SYKORA, Zbyněk PROKOP, A. GHOSE, David BEDNÁŘ et. al.

Basic information

Original name

Molecular Gating of an Engineered Enzyme Captured in Real Time

Authors

KOKKONEN, Piia Pauliina (246 Finland, belonging to the institution), J. SYKORA (203 Czech Republic), Zbyněk PROKOP (203 Czech Republic, belonging to the institution), A. GHOSE (356 India), David BEDNÁŘ (203 Czech Republic, belonging to the institution), M. AMARO (620 Portugal), Koen BEERENS (56 Belgium, belonging to the institution), Šárka NEVOLOVÁ (203 Czech Republic, belonging to the institution), Michaela SLÁNSKÁ (203 Czech Republic, belonging to the institution), Jan BREZOVSKÝ (203 Czech Republic, belonging to the institution), Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution) and M. HOF (276 Germany)

Edition

Journal of the American Chemical Society, Washington, American Chemical Society, 2018, 0002-7863

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10401 Organic chemistry

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 14.695

RIV identification code

RIV/00216224:14310/18:00101750

Organization unit

Faculty of Science

DOI

UT WoS

000454751800028

Keywords in English

PHOTOINDUCED ELECTRON-TRANSFER; CONFORMATIONAL DYNAMICS; CATALYTIC MECHANISM; PROTEIN DYNAMICS; NUCLEIC-ACIDS; ACTIVE-SITE; FORCE-FIELD; EVOLUTION; MOTIONS; SIMULATION
Změněno: 23/4/2024 14:18, Mgr. Michal Petr

Abstract

V originále

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.

Links

CZ.02.1.01/0.0/0.0/16_013/0001761, interní kód MU
Name: RECETOX RI - OP VVV (Acronym: RECETOX RI)
Investor: Ministry of Education, Youth and Sports of the CR, Priority axis 1: Strengthening capacities for high-quality research
GA16-06096S, research and development project
Name: Objasnění významu dynamických tunelů pro enzymatickou katalýzu: simulace a fluorescenční experimenty
Investor: Czech Science Foundation
GA16-07965S, research and development project
Name: Řízená evoluce dynamických elementů v enzymech s využitím mikrofluidních čipů
Investor: Czech Science Foundation
LM2015047, research and development project
Name: Česká národní infrastruktura pro biologická data (Acronym: ELIXIR-CZ)
Investor: Ministry of Education, Youth and Sports of the CR, Czech National Infrastructure for Biological Data
LM2015051, research and development project
Name: Centrum pro výzkum toxických látek v prostředí (Acronym: RECETOX RI)
Investor: Ministry of Education, Youth and Sports of the CR
LM2015055, research and development project
Name: Centrum pro systémovou biologii (Acronym: C4SYS)
Investor: Ministry of Education, Youth and Sports of the CR