Insights from molecular dynamics simulations into pH-dependent enantioselective hydrolysis of ibuprofen esters by Candida rugosa lipase.

An interesting observation was found during our continued studies on the hydrolysis of ibuprofen esters by Candida rugosa lipase (CRL). An important role is played by pH in the stereospecific hydrolysis of these esters. The flap region of CRL plays a significant role in the access of the substrate to the active site of the enzyme. At pH 5.6, 48% of the methyl ester and 5% of the butyl ester of ibuprofen were hydrolysed in 5.5 h, whereas at pH 7.2, 9% of methyl ester and 45% of the butyl ester of ibuprofen was hydrolysed in a identical reaction time using CRL. This lead us to assume that CRL prefers the methyl ester of ibuprofen as a substrate at an acidic pH and the butyl ester of ibuprofen at a neutral pH. Therefore, in order to understand the role of pH in the substrate selection by CRL for the esters of ibuprofen we used the crystallographic coordinates of the open form of the CRL (1CRL) for molecular dynamics (MD) simulations under acidic and neutral conditions for 2 ns using GROMACS. The final structures obtained after simulation in acidic and neutral conditions were compared with the energy-minimized structure, and the root-mean-square deviations (r.m.s.ds) were calculated. The r.m.s.d. of the CRL flap at neutral pH was found to be greater than that of the CRL flap at acidic pH. The extent to which the flap opens at neutral pH allowed the bulkier substrate, the butyl ester of ibuprofen, to diffuse into the active site and provides the best enzyme-substrate fit for this specific substrate. At acidic pH there is a decreased opening of the flap thereby accommodating a more compact substrate, namely the methyl ester of ibuprofen. Thus, simulation experiments using MD provide reasonable insight for the pH-dependent substrate selectivity of CRL in aqueous environments.