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Molecular dynamics derived life times of active substrate binding poses explain K(M) of laccase mutants

Fungal laccases (EC 1.10.3.2) are important multi-copper oxidases with broad substrate specificity. Laccases from Trametes versicolor (TvL) are among the best-characterized of these enzymes. Mutations in the substrate-binding site of TvL substantially affect K(M), but a molecular understanding of th...

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Autores principales: Mehra, Rukmankesh, Meyer, Anne S., Kepp, Kasper P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9089231/
https://www.ncbi.nlm.nih.gov/pubmed/35558910
http://dx.doi.org/10.1039/c8ra07138a
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author Mehra, Rukmankesh
Meyer, Anne S.
Kepp, Kasper P.
author_facet Mehra, Rukmankesh
Meyer, Anne S.
Kepp, Kasper P.
author_sort Mehra, Rukmankesh
collection PubMed
description Fungal laccases (EC 1.10.3.2) are important multi-copper oxidases with broad substrate specificity. Laccases from Trametes versicolor (TvL) are among the best-characterized of these enzymes. Mutations in the substrate-binding site of TvL substantially affect K(M), but a molecular understanding of this effect is missing. We explored the effect of TvL mutations on K(M) for the standard laccase substrate 2,6-dimethoxyphenol using 4500 ns of molecular dynamics, docking, and MMGBSA free energy computations. We show that changes in K(M) due to mutation consistently correlate with the dynamics of the substrates within the substrate-binding site. We find that K(M) depends on the lifetime (“dynamic stability”) of the enzyme-substrate complex as commonly assumed. We then further show that MMGBSA-derived free energies of substrate binding in the active pose consistently reproduce large vs. small experimental K(M) values. Our results indicate that hydrophobic packing of the substrate near the T1 binding site of the laccase is instrumental for high turnover via K(M). We also address the more general question of how enzymes such as laccases gain advantage of lower K(M) despite the Sabatier principle, which disfavors a stable enzyme–substrate complex. Our data suggest that the observed K(M) relates directly to the lifetime of the active substrate pose within a protein. In contrast, the thermochemical stability of the enzyme–substrate complex reflects an ensemble average of all enzyme–substrate binding poses. This distinction may explain how enzymes work by favoring longer residence time in the active pose without too favorable general enzyme–substrate interactions, a principle that may aid the rational design of enzymes.
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spelling pubmed-90892312022-05-11 Molecular dynamics derived life times of active substrate binding poses explain K(M) of laccase mutants Mehra, Rukmankesh Meyer, Anne S. Kepp, Kasper P. RSC Adv Chemistry Fungal laccases (EC 1.10.3.2) are important multi-copper oxidases with broad substrate specificity. Laccases from Trametes versicolor (TvL) are among the best-characterized of these enzymes. Mutations in the substrate-binding site of TvL substantially affect K(M), but a molecular understanding of this effect is missing. We explored the effect of TvL mutations on K(M) for the standard laccase substrate 2,6-dimethoxyphenol using 4500 ns of molecular dynamics, docking, and MMGBSA free energy computations. We show that changes in K(M) due to mutation consistently correlate with the dynamics of the substrates within the substrate-binding site. We find that K(M) depends on the lifetime (“dynamic stability”) of the enzyme-substrate complex as commonly assumed. We then further show that MMGBSA-derived free energies of substrate binding in the active pose consistently reproduce large vs. small experimental K(M) values. Our results indicate that hydrophobic packing of the substrate near the T1 binding site of the laccase is instrumental for high turnover via K(M). We also address the more general question of how enzymes such as laccases gain advantage of lower K(M) despite the Sabatier principle, which disfavors a stable enzyme–substrate complex. Our data suggest that the observed K(M) relates directly to the lifetime of the active substrate pose within a protein. In contrast, the thermochemical stability of the enzyme–substrate complex reflects an ensemble average of all enzyme–substrate binding poses. This distinction may explain how enzymes work by favoring longer residence time in the active pose without too favorable general enzyme–substrate interactions, a principle that may aid the rational design of enzymes. The Royal Society of Chemistry 2018-11-01 /pmc/articles/PMC9089231/ /pubmed/35558910 http://dx.doi.org/10.1039/c8ra07138a Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/
spellingShingle Chemistry
Mehra, Rukmankesh
Meyer, Anne S.
Kepp, Kasper P.
Molecular dynamics derived life times of active substrate binding poses explain K(M) of laccase mutants
title Molecular dynamics derived life times of active substrate binding poses explain K(M) of laccase mutants
title_full Molecular dynamics derived life times of active substrate binding poses explain K(M) of laccase mutants
title_fullStr Molecular dynamics derived life times of active substrate binding poses explain K(M) of laccase mutants
title_full_unstemmed Molecular dynamics derived life times of active substrate binding poses explain K(M) of laccase mutants
title_short Molecular dynamics derived life times of active substrate binding poses explain K(M) of laccase mutants
title_sort molecular dynamics derived life times of active substrate binding poses explain k(m) of laccase mutants
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9089231/
https://www.ncbi.nlm.nih.gov/pubmed/35558910
http://dx.doi.org/10.1039/c8ra07138a
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