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

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 tu...

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Autores principales: Kokkonen, Piia, Slanska, Michaela, Dockalova, Veronika, Pinto, Gaspar P., Sánchez-Carnerero, Esther M., Damborsky, Jiri, Klán, Petr, Prokop, Zbynek, Bednar, David
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Research Network of Computational and Structural Biotechnology 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152659/
https://www.ncbi.nlm.nih.gov/pubmed/32308927
http://dx.doi.org/10.1016/j.csbj.2020.03.017
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author Kokkonen, Piia
Slanska, Michaela
Dockalova, Veronika
Pinto, Gaspar P.
Sánchez-Carnerero, Esther M.
Damborsky, Jiri
Klán, Petr
Prokop, Zbynek
Bednar, David
author_facet Kokkonen, Piia
Slanska, Michaela
Dockalova, Veronika
Pinto, Gaspar P.
Sánchez-Carnerero, Esther M.
Damborsky, Jiri
Klán, Petr
Prokop, Zbynek
Bednar, David
author_sort Kokkonen, Piia
collection PubMed
description 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 + I211L 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 + I211L. 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.
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spelling pubmed-71526592020-04-17 The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step Kokkonen, Piia Slanska, Michaela Dockalova, Veronika Pinto, Gaspar P. Sánchez-Carnerero, Esther M. Damborsky, Jiri Klán, Petr Prokop, Zbynek Bednar, David Comput Struct Biotechnol J Research Article 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 + I211L 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 + I211L. 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. Research Network of Computational and Structural Biotechnology 2020-03-25 /pmc/articles/PMC7152659/ /pubmed/32308927 http://dx.doi.org/10.1016/j.csbj.2020.03.017 Text en © 2020 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Kokkonen, Piia
Slanska, Michaela
Dockalova, Veronika
Pinto, Gaspar P.
Sánchez-Carnerero, Esther M.
Damborsky, Jiri
Klán, Petr
Prokop, Zbynek
Bednar, David
The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step
title The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step
title_full The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step
title_fullStr The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step
title_full_unstemmed The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step
title_short The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step
title_sort impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152659/
https://www.ncbi.nlm.nih.gov/pubmed/32308927
http://dx.doi.org/10.1016/j.csbj.2020.03.017
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