Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents
Language English Country Germany Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- 2-Propanol chemistry metabolism MeSH
- Acetone chemistry metabolism MeSH
- Time Factors MeSH
- Spectrometry, Fluorescence MeSH
- Formamides chemistry metabolism MeSH
- Hydrolases chemistry metabolism MeSH
- Catalytic Domain MeSH
- Kinetics MeSH
- Solvents chemistry metabolism MeSH
- Molecular Dynamics Simulation MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- 2-Propanol MeSH
- Acetone MeSH
- formamide MeSH Browser
- Formamides MeSH
- haloalkane dehalogenase MeSH Browser
- Hydrolases MeSH
- Solvents MeSH
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 K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). 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.
References provided by Crossref.org
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