These authors contributed equally to this work.
Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
Article first published online: 3 DEC 2004
Copyright © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Volume 6, Issue 2, pages 331–336, February 4, 2005
How to Cite
Carlqvist, P., Svedendahl, M., Branneby, C., Hult, K., Brinck, T. and Berglund, P. (2005), Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions. ChemBioChem, 6: 331–336. doi: 10.1002/cbic.200400213
- Issue published online: 28 JAN 2005
- Article first published online: 3 DEC 2004
- Manuscript Received: 24 JUN 2004
- enzyme catalysis;
- Michael addition;
- protein design;
- quantum chemistry
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 kcatvalues were found to be in the range of 10−3to 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.