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@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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