A model of the pressure dependence of the enantio selectivity of Candida rugosa lipase towards (+/-)-menthol

Transesterification of (+/-)-menthol using propionic acid anhydride and Can dida rugosa lipase was performed in chloroform and water at different press ures (1, 10, 50, and 100 bar) to study the pressure dependence of enantiose lectivity E. As a result, E significantly decreased with increasing pressur e from E = 55 (1 bar) to E = 47 (10 bar), E = 37 (50 bar), and E = 9 (100 b ar). To rationalize the experimental findings, molecular dynamics simulatio ns 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 a nd the catalytic H449. In the crystal structure of the Candida rugosa lipas e, this cavity is filled with six water molecules. The number of water mole cules in this cavity gradually increased with increasing pressure: six mole cules in the simulation at 1 bar, 10 molecules at 10 bar, 12 molecules at 5 0 bar, and 13 molecules at 100 bar. Likewise, the volume of the cavity prog ressively increased from about 1864 Angstrom (3) in the simulation at 1 bar to 2529 Angstrom (3) at 10 bar, 2526 Angstrom (3) at 50 bar, and 2617 Angs trom (3) at 100 bar. At 100 bar, one water molecule slipped between F415 an d H449, displacing the catalytic histidine side chain and thus opening the cavity to form a continuous water channel. The rotation of the side chain l eads 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 th e nonpreferred enantiomer gets gradually more reactive. This observation co rrelates with the gradually decreasing enantioselectivity E.