Exploring the active-site of a rationally redesigned lipase for catalysis of Michael-type additions.
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 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 kca 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.
Carlqvist
Peter
P
Physical Chemistry, Royal Institute of Technology (KTH), 100 44 Stockholm, Sweden.
Svedendahl
Maria
M
Branneby
Cecilia
C
Hult
Karl
K
Brinck
Tore
T
Berglund
Per
P
D001665:Binding Sites; D005656:Fungal Proteins; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013816:Thermodynamics
D005656:Fungal Proteins; D012997:Solvents; D013438:Sulfhydryl Compounds; D008049:Lipase; C431349:lipase B, Candida antarctica