Expansion of Access Tunnels and Active-Site Cavities Influence Activity of Haloalkane Dehalogenases in Organic Cosolvents

Authors

  • Veronika Stepankova,

    1. Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5A13, 625 00 Brno (Czech Republic)
    2. International Clinical Research Center, St. Anne's University Hospital, 656 91 Brno (Czech Republic)
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  • Dr. Morteza Khabiri,

    1. Institute of Nanobiology and Structural Biology, Global Change Research Center, Academy of Sciences of the Czech Republic, 373 33 Nove Hrady (Czech Republic)
    2. Faculty of Sciences, University of South Bohemia, 373 33 Nove Hrady (Czech Republic)
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  • Dr. Jan Brezovsky,

    1. Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5A13, 625 00 Brno (Czech Republic)
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  • Antonin Pavelka,

    1. Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5A13, 625 00 Brno (Czech Republic)
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  • Dr. Jan Sykora,

    1. J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, 182 23 Praha (Czech Republic)
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  • Dr. Mariana Amaro,

    1. J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, 182 23 Praha (Czech Republic)
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  • Dr. Babak Minofar,

    1. Institute of Nanobiology and Structural Biology, Global Change Research Center, Academy of Sciences of the Czech Republic, 373 33 Nove Hrady (Czech Republic)
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  • Assoc. Prof. Zbynek Prokop,

    1. Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5A13, 625 00 Brno (Czech Republic)
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  • Prof. Martin Hof,

    1. J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, 182 23 Praha (Czech Republic)
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  • Assoc. Prof. Rudiger Ettrich,

    1. Institute of Nanobiology and Structural Biology, Global Change Research Center, Academy of Sciences of the Czech Republic, 373 33 Nove Hrady (Czech Republic)
    2. Faculty of Sciences, University of South Bohemia, 373 33 Nove Hrady (Czech Republic)
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  • Dr. Radka Chaloupkova,

    Corresponding author
    1. Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5A13, 625 00 Brno (Czech Republic)
    • Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5A13, 625 00 Brno (Czech Republic)

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  • Prof. Jiri Damborsky

    Corresponding author
    1. Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5A13, 625 00 Brno (Czech Republic)
    2. International Clinical Research Center, St. Anne's University Hospital, 656 91 Brno (Czech Republic)
    • Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5A13, 625 00 Brno (Czech Republic)

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Abstract

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein–solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme–solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K0.5 and increasing kcat, or caused enzyme inactivation by promoting substrate inhibition and decreasing kcat. The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein–solvent pair and effective cosolvent concentration.

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