Mechanism of enantioselectivity of lipases and other synthetically useful hydrolases

Lipases are biocatalysts most widely used in organic synthesis. Unusual but attractive feature of lipases is that, in addition to high catalytic activity and thermostability in organic solvents, lipases show high enantioselectivity and broad substrate specificity simultaneously. They show excellent enantioselectivity especially toward a wide range of secondary alcohols. The mechanistic details of stereoselective organic reactions are relatively well understood, and knowledge has been used to develop new chemical reagents. In contrast, biocatalysts are behind chemical reagents in rational design approaches, which is partly due to the mechanistic ambiguity of enzymatic reactions. The mechanistic aspects of enantioselective biocatalysts are nevertheless becoming clear. This review provides an overview of the studies aimed at understanding the mechanisms of enantioselectivity of synthetically useful hydrolases Such as lipases, subtilisins and chymotrypsins toward unnatural chiral substrates. Several methods for addressing the mechanism are introduced: (i) substrate mapping, (ii) X-ray crystallographic analysis, (iii) computational calculations, (iv) kinetic analysis, (v) thermodynamic analysis, (vi) site-directed or random mutagenesis, (vii) spectroscopic methods Such as fluorescence, ESR, and mass spectroscopy. Different models and mechanisms proposed so far are selected and explained. The chemical principles revealed by the mechanistic studies will be useful for (i) using the enzymes in organic synthesis efficiently, (ii) altering the features of the enzymes rationally, (iii) utilizing them as a tool for determining the absolute stereochemistry of molecules, and (iv) designing new artificial catalysts mimicking the catalytic machinery of the enzymes.