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An understanding of protein\u2013solvent 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\u2010state kinetics assays, molecular dynamics simulations, and time\u2010resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme\u2013solvent 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 <jats:italic>K<\/jats:italic><jats:sub>0.5<\/jats:sub> and increasing <jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>, or caused enzyme inactivation by promoting substrate inhibition and decreasing <jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>. The induced activation and inhibition of the enzymes correlated with expansion of the active\u2010site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active\u2010sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein\u2013solvent pair and effective cosolvent concentration.<\/jats:p>","DOI":"10.1002\/cbic.201200733","type":"journal-article","created":{"date-parts":[[2013,4,5]],"date-time":"2013-04-05T14:20:27Z","timestamp":1365171627000},"page":"890-897","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":45,"title":["Expansion of Access Tunnels and Active\u2010Site Cavities Influence Activity of Haloalkane Dehalogenases in Organic Cosolvents"],"prefix":"10.1002","volume":"14","author":[{"given":"Veronika","family":"Stepankova","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Morteza","family":"Khabiri","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jan","family":"Brezovsky","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Antonin","family":"Pavelka","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jan","family":"Sykora","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mariana","family":"Amaro","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Babak","family":"Minofar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zbynek","family":"Prokop","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martin","family":"Hof","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rudiger","family":"Ettrich","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Radka","family":"Chaloupkova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiri","family":"Damborsky","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2013,4,5]]},"reference":[{"key":"e_1_2_7_1_2","doi-asserted-by":"publisher","DOI":"10.1002\/1521-3757(20000703)112:13<2312::AID-ANGE2312>3.0.CO;2-4"},{"key":"e_1_2_7_1_3","doi-asserted-by":"publisher","DOI":"10.1002\/1521-3773(20000703)39:13<2226::AID-ANIE2226>3.0.CO;2-L"},{"key":"e_1_2_7_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/35051719"},{"key":"e_1_2_7_3_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0601113103"},{"key":"e_1_2_7_4_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.82.10.3192"},{"key":"e_1_2_7_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.6729453"},{"key":"e_1_2_7_6_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.2658059"},{"key":"e_1_2_7_7_2","doi-asserted-by":"publisher","DOI":"10.1021\/ja970629a"},{"key":"e_1_2_7_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0167-7799(97)01013-5"},{"key":"e_1_2_7_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/35051736"},{"key":"e_1_2_7_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbapap.2007.05.013"},{"key":"e_1_2_7_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2007.10.007"},{"key":"e_1_2_7_12_2","doi-asserted-by":"publisher","DOI":"10.1006\/bbrc.2001.4282"},{"key":"e_1_2_7_13_2","doi-asserted-by":"publisher","DOI":"10.1023\/A:1020530220774"},{"key":"e_1_2_7_14_2","doi-asserted-by":"publisher","DOI":"10.1271\/bbb.67.1660"},{"key":"e_1_2_7_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.enzmictec.2004.09.014"},{"key":"e_1_2_7_16_2","doi-asserted-by":"publisher","DOI":"10.1002\/bip.20577"},{"key":"e_1_2_7_17_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2011.01.088"},{"key":"e_1_2_7_18_2","doi-asserted-by":"publisher","DOI":"10.2174\/138920308785132686"},{"key":"e_1_2_7_19_2","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/gzn048"},{"key":"e_1_2_7_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cbpa.2004.02.012"},{"key":"e_1_2_7_21_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.71.8.4372-4379.2005"},{"key":"e_1_2_7_22_2","doi-asserted-by":"publisher","DOI":"10.1002\/prot.21313"},{"key":"e_1_2_7_23_2","doi-asserted-by":"crossref","first-page":"6247","DOI":"10.1002\/ange.201001753","volume":"122","author":"Prokop Z.","year":"2010","journal-title":"Angew. 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