A structure-based rationalization of the enantiopreference of subtilisin toward secondary alcohols and isosteric primary amines

Lipases favor one enantiomer of secondary alcohols (HOCHRR') and isosteric primary amines (NH₂CHRR'), while subtilisin favors the other enantiomer. In both cases, simple rules based on the size of the substituents at the stereocenter predict which enantiomer reacts faster. Thus, lipases and subtilisin are a pair of complementary enantioselective reagents for organic synthesis. The success of these rules suggests that these hydrolases distinguish between enantiomer primarily by the size of the substituents. Previously, we proposed a molecular mechanism for the enantiopreference of lipases based on the X-ray crystal structure of transition state analogs bound to a lipase. Here we suggest that a similar mechanism can also account for the opposite enantiopreference of subtilisin. The catalytic machinery (catalytic triad plus the oxyanion-stabilizing residues) in lipases is approximately the mirror image of that in subtilisin. In both hydrolases, the protein fold, as it assembles the catalytic machinery, also creates a restricted pocket for one substituent in the substrate ('M' or medium- sized). However, the catalytic His residue lies on opposite sides of this pocket in the two hydrolases. We propose that enantioselection arises from (1) the limited size of this pocket, (2) and a required hydrogen bond between the catalytic His and the oxygen or nitrogen of the alcohol or amine. This mechanism for enantioselection differs from that proposed by Derewenda and Wei who focussed on which carbonyl face in the ester or amide is attacked. Lipases and subtilisin indeed attack opposite faces, but we propose that this difference does not set the enantiopreference toward secondary alcohols.