Exploring the Active‐Site of a Rationally Redesigned Lipase for Catalysis of Michael‐Type Additions

• Published in: ChemBioChem
• Main Authors: Carlqvist, Peter, Svedendahl, Maria, Branneby, Cecilia, Hult, Karl, Brinck, Tore, Berglund, Per
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2005
• Subjects: Antarc*; Antarctica
• Online Access: http://dx.doi.org/10.1002/cbic.200400213; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fcbic.200400213; https://onlinelibrary.wiley.com/doi/full/10.1002/cbic.200400213

Abstract Michael‐type additions of various thiols and α,β‐unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild‐type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant lacks 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.