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

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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 kcat 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 107. Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.