The impact of tunnel mutations on enzymatic catalysis depends
on the tunnel-substrate complementarity and the rate-limiting
step

J 2020

The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step

KOKKONEN, Piia Pauliina, Michaela SLÁNSKÁ, Veronika DOČKALOVÁ, José Gaspar RANGEL PAMPLONA PIZARRO PINTO, Esther Maria MARQUEZ SANCHEZ - CARNERERO et. al.

Basic information

Original name

The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step

Authors

KOKKONEN, Piia Pauliina (246 Finland, belonging to the institution), Michaela SLÁNSKÁ (203 Czech Republic, belonging to the institution), Veronika DOČKALOVÁ (203 Czech Republic, belonging to the institution), José Gaspar RANGEL PAMPLONA PIZARRO PINTO (620 Portugal, belonging to the institution), Esther Maria MARQUEZ SANCHEZ - CARNERERO (724 Spain, belonging to the institution), Jiří DAMBORSKÝ (203 Czech Republic, belonging to the institution), Petr KLÁN (203 Czech Republic, belonging to the institution), Zbyněk PROKOP (203 Czech Republic, belonging to the institution) and David BEDNÁŘ (203 Czech Republic, guarantor, belonging to the institution)

Edition

Computational and Structural Biotechnology Journal, Amsterdam, Elsevier, 2020, 2001-0370

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10608 Biochemistry and molecular biology

Country of publisher

Netherlands

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 7.271

RIV identification code

RIV/00216224:14310/20:00118046

Organization unit

Faculty of Science

DOI

http://dx.doi.org/10.1016/j.csbj.2020.03.017

UT WoS

000607729500006

Keywords in English

Enzyme kinetics; Enzyme mutation; Substrate specificity

Abstract

V originále

Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tunnel of haloalkane dehalogenase LinB have been previously engineered for improved dehalogenation of 1,2-dibromoethane (DBE). The first chemical step of DBE conversion was enhanced by L177W mutation in the main tunnel, but the rate-limiting product release was slowed down because the mutation blocked the main access tunnel and hindered protein dynamics. Three additional mutations W140A + F143L + 1211L opened-up the auxiliary tunnel and enhanced the product release, making this four-point variant the most efficient catalyst with DBE. Here we study the impact of these mutations on the catalysis of bulky aromatic substrates, 4-(bromomethyl)-6,7-dimethoxycoumarin (COU) and 8-chloromethyl-4,4'-difluoro-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (BDP). The rate-limiting step of DBE conversion is the product release, whereas the catalysis of COU and BDP is limited by the chemical step. The catalysis of COU is mainly impaired by the mutation L177W, whereas the conversion of BDP is affected primarily by the mutations W140A + F143L +1211L. The combined computational and kinetic analyses explain the differences in activities between the enzyme-substrate pairs. The effect of tunnel mutations on catalysis depends on the rate-limiting step, the complementarity of the tunnels with the substrates and is clearly specific for each enzyme-substrate pair.

Links

EF16_013/0001761, research and development project

Name: RECETOX RI

EF17_050/0008496, research and development project

Name: MSCAfellow@MUNI

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

LM2015085, research and development project

Name: CERIT Scientific Cloud (Acronym: CERIT-SC)

Investor: Ministry of Education, Youth and Sports of the CR, CERIT Scientific Cloud

LM2018140, research and development project

Name: e-Infrastruktura CZ (Acronym: e-INFRA CZ)

Investor: Ministry of Education, Youth and Sports of the CR

90042, large research infrastructures

Name: CESNET II

Citovat

KOKKONEN, Piia Pauliina, Michaela SLÁNSKÁ, Veronika DOČKALOVÁ, José Gaspar RANGEL PAMPLONA PIZARRO PINTO, Esther Maria MARQUEZ SANCHEZ - CARNERERO, Jiří DAMBORSKÝ, Petr KLÁN, Zbyněk PROKOP and David BEDNÁŘ. The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step. Computational and Structural Biotechnology Journal. Amsterdam: Elsevier, 2020, vol. 18, No 2020, p. 805-813. ISSN 2001-0370. Available from: https://dx.doi.org/10.1016/j.csbj.2020.03.017.

@article{1734897,
   author = {Kokkonen, Piia Pauliina and Slánská, Michaela and Dočkalová, Veronika and Rangel Pamplona Pizarro Pinto, José Gaspar and Marquez Sanchez and Carnerero, Esther Maria and Damborský, Jiří and Klán, Petr and Prokop, Zbyněk and Bednář, David},
   article_location = {Amsterdam},
   article_number = {2020},
   doi = {http://dx.doi.org/10.1016/j.csbj.2020.03.017},
   keywords = {Enzyme kinetics; Enzyme mutation; Substrate specificity},
   language = {eng},
   issn = {2001-0370},
   journal = {Computational and Structural Biotechnology Journal},
   title = {The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step},
   url = {https://doi.org/10.1016/j.csbj.2020.03.017},
   volume = {18},
   year = {2020}
}

TY  - JOUR
ID  - 1734897
AU  - Kokkonen, Piia Pauliina - Slánská, Michaela - Dočkalová, Veronika - Rangel Pamplona Pizarro Pinto, José Gaspar - Marquez Sanchez - Carnerero, Esther Maria - Damborský, Jiří - Klán, Petr - Prokop, Zbyněk - Bednář, David
PY  - 2020
TI  - The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step
JF  - Computational and Structural Biotechnology Journal
VL  - 18
IS  - 2020
SP  - 805-813
EP  - 805-813
PB  - Elsevier
SN  - 20010370
KW  - Enzyme kinetics
KW  - Enzyme mutation
KW  - Substrate specificity
UR  - https://doi.org/10.1016/j.csbj.2020.03.017
L2  - https://doi.org/10.1016/j.csbj.2020.03.017
N2  - Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tunnel of haloalkane dehalogenase LinB have been previously engineered for improved dehalogenation of 1,2-dibromoethane (DBE). The first chemical step of DBE conversion was enhanced by L177W mutation in the main tunnel, but the rate-limiting product release was slowed down because the mutation blocked the main access tunnel and hindered protein dynamics. Three additional mutations W140A + F143L + 1211L opened-up the auxiliary tunnel and enhanced the product release, making this four-point variant the most efficient catalyst with DBE. Here we study the impact of these mutations on the catalysis of bulky aromatic substrates, 4-(bromomethyl)-6,7-dimethoxycoumarin (COU) and 8-chloromethyl-4,4'-difluoro-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (BDP). The rate-limiting step of DBE conversion is the product release, whereas the catalysis of COU and BDP is limited by the chemical step. The catalysis of COU is mainly impaired by the mutation L177W, whereas the conversion of BDP is affected primarily by the mutations W140A + F143L +1211L. The combined computational and kinetic analyses explain the differences in activities between the enzyme-substrate pairs. The effect of tunnel mutations on catalysis depends on the rate-limiting step, the complementarity of the tunnels with the substrates and is clearly specific for each enzyme-substrate pair.
ER  -

KOKKONEN, Piia Pauliina, Michaela SLÁNSKÁ, Veronika DOČKALOVÁ, José Gaspar RANGEL PAMPLONA PIZARRO PINTO, Esther Maria MARQUEZ SANCHEZ - CARNERERO, Jiří DAMBORSKÝ, Petr KLÁN, Zbyněk PROKOP and David BEDNÁŘ. The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step. \textit{Computational and Structural Biotechnology Journal}. Amsterdam: Elsevier, 2020, vol.~18, No~2020, p.~805-813. ISSN~2001-0370. Available from: https://dx.doi.org/10.1016/j.csbj.2020.03.017.

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