A model of the pressure dependence of the enantioselectivity of Candida rugosalipase towards (+/-)-menthol.
Transesterification of (+/-)-menthol using propionic acid anhydride and Candida rugosa lipase was performed in chloroform and water at different pressures (1, 10, 50, and 100 bar) to study the pressure dependence of enantioselectivity E. As a result, E significantly decreased with increasing pressure from E = 55 (1 bar) to E = 47 (10 bar), E = 37 (50 bar), and E = 9 (100 bar). To rationalize the experimental findings, molecular dynamics simulations of Candida rugosa lipase were carried out. Analyzing the lipase geometry at 1, 10, 50, and 100 bar revealed a cavity in the Candida rugosa lipase. The cavity leads from a position on the surface distinct from the substrate binding site to the core towards the active site, and is limited by F415 and the catalytic H449. In the crystal structure of the Candida rugosa lipase, this cavity is filled with six water molecules. The number of water molecules in this cavity gradually increased with increasing pressure: six molecules in the simulation at 1 bar, 10 molecules at 10 bar, 12 molecules at 50 bar, and 13 molecules at 100 bar. Likewise, the volume of the cavity progressively increased from about 1864 A(3) in the simulation at 1 bar to 2529 A(3) at 10 bar, 2526 A(3) at 50 bar, and 2617 A(3) at 100 bar. At 100 bar, one water molecule slipped between F415 and H449, displacing the catalytic histidine side chain and thus opening the cavity to form a continuous water channel. The rotation of the side chain leads to a decreased distance between the H449-N epsilon and the (+)-menthyl-oxygen (nonpreferred enantiomer) in the acyl enzyme intermediate, a factor determining the enantioselectivity of the lipase. Although the geometry of the preferred enantiomer is similar in all simulations, the geometry of the nonpreferred enantiomer gets gradually more reactive. This observation correlates with the gradually decreasing enantioselectivity E.
Kahlow
U H
UH
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Schmid
R D
RD
Pleiss
J
J
D001711:Biotransformation; D002175:Candida / Q000201:enzymology*; D003198:Computer Simulation; D008049:Lipase / Q000378:metabolism*; D008610:Menthol / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D011312:Pressure; D011487:Protein Conformation; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry
D014867:Water; D008610:Menthol; D008049:Lipase