Rhizopus oryzae lipase-catalyzed stereospecific esterification of 2-monoradylglycerols - a comparison to corresponding triradylglycerol hydrolysis

In a model elaborated earlier to understand and predict the stereopreferenc e of Rhizopus oryzae lipase (ROL) catalyzed hydrolysis of triradylglycerols , we identified the degree of flexibility of the C1'-X' bond (X = O for eth er, N for amide, C for alkyl, methylene, and a phenylring, respectively) ad jacent to C2 of glycerol being responsible for the discrimination of the en antiomers (Kovac et al., fur. J. Lipid Sci. Technol. 2000, 61-72). During c atalysis of forward and back reaction - hydrolysis and esterification in ei ther case the carbonyl carbon of the sn-1 or sn-3 fatty acid binds to the a ctive site serine of ROL leading to a covalently bound intermediate, which was simulated in the model. Thus, we assumed that stereoselectivity of ROL in esterification of corresponding 2-monoradylglycerols with oleic acid in cyclohexane should follow the same model. As predicted by this model 2-mono radylglycerols with "rigid" phenyl and amide substituents were esterified a t the sn-3 position, and those with "flexible" ether substituents at the sn -1 position. However, enantiomeric excess of wild type ROL in esterifying 2 -monaradylglycerols with flexible benzylether and methylene substituents di ffered by around 50% as compared to hydrolysis experiments with correspondi ng triradylglycerols. In addition esterification of 2-monoradylglycerol wit h flexible ether substituent by ROL L258F/L254F double mutant was essential ly non-selective compared to corresponding triradylglycerol where enantiome ric excess was 58% sn-1. Whether water activity was a factor determining th ese discrepancies was investigated for ROL- and double mutant enzyme-cataly zed esterification of the ether and methylene substrates under controlled w ater activities from 0.02-0.85. In all cases stereoselectivities observed w ere independent from water activities. In conclusion, the model describing the stereoselective course of aqueous hydrolysis of triradylglycerols catal yzed by ROL in most cases applies to the esterification reaction in organic solvent. Differences in stereoselectivity observed are attributed to reduc ed possibilities for interaction of 2-monoradylglycerol substrates with the binding sites of ROL as compared to those of triradylglycerol substrates.