Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
2005 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, 331-336 p.Article in journal (Refereed) Published
Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant locks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The k(cat) values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.
Place, publisher, year, edition, pages
2005. Vol. 6, 331-336 p.
DENSITY-FUNCTIONAL THEORY; ENZYMATIC-REACTIONS; HYDROLYTIC ENZYMES; ALKALINE PROTEASE; BACILLUS-SUBTILIS; ORGANIC MEDIA; GROUND-STATE; MECHANISM
IdentifiersURN: urn:nbn:se:kth:diva-5113DOI: 10.1002/cbic.200400213ISI: 000226957100014ScopusID: 2-s2.0-20544452621OAI: oai:DiVA.org:kth-5113DiVA: diva2:7880