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

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Publikace nespadá pod Fakultu sportovních studií, ale pod Přírodovědeckou fakultu. Oficiální stránka publikace je na webu muni.cz.
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ŠTĚPÁNKOVÁ Veronika KHABIRI M. BREZOVSKÝ Jan PAVELKA Antonín SYKORA J. AMARO M. MINOFAR B. PROKOP Zbyněk HOF M. ETTRICH R. CHALOUPKOVÁ Radka DAMBORSKÝ Jiří

Rok publikování 2013
Druh Článek v odborném periodiku
Časopis / Zdroj ChemBioChem
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
Doi http://dx.doi.org/10.1002/cbic.201200733
Obor Biochemie
Klíčová slova haloalkane dehalogenases
Popis 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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