Structural basis of stereoselectivity in Candida rugosa lipase-catalyzed hydrolysis of secondary alcohols

Abstract.

Lipases are widely used catalysts for highly enantioselective resolution of chiral secondary alcohols. While stereopreference is determined predominantly by the substrate structure, stereoselectivity (enantioselectivity and diastereoselectivity) depends on the atomic details of interactions between substrate and lipase. Experimentally obtained stereoselectivity and activity in the hydrolysis of butanoic acid esters of two secondary alcohols with two neighboring stereocenters by Candida rugosa lipase have been investigated by computer-aided molecular modeling of tetrahedral substrate intermediates in complex with the lipase. Breakdown of these intermediates is considered to be the rate-limiting step. Steric interactions of stereoisomers with the side chain of catalytic histidine led to different orientations of the imidazole. The distance d(H_{N ε}–O_alc) between H_{N ε} of the imidazole side chain of catalytic histidine and the alcohol oxygen of the substrate was identified to correlate with the experimentally determined reactivity order of the four stereoisomers. Modeled distances d(H_{N ε}–O_alc) were short (=1.8 Å) for RR stereoisomers, which were also found to be hydrolyzed most rapidly experimentally; distances d(H_{N ε}–O_alc) were about 2 Å for SS and SR stereoisomers, which were converted at similar rates but at a lower rate than RR stereoisomers; finally, distances d(H_{N ε}–O_alc) for SR stereoisomers were greater than 4 Å, in accordance with very slow conversion of SR stereoisomers.