Differences in Binding Modes of Enantiomers of 1-Acetamido Boronic Acid Based Protease Inhibitors: Crystal Structures of γ-Chymotrypsin and Subtilisin Carlsberg Complexes,

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Abstract
In order to probe the structural basis of stereoselectivity in the serine protease family, a series of enantiomeric boronic acids RCH2CH(NHCOCH3)B(OH)2 has been synthesized and kinetically characterized as transition-state analog inhibitors using α-chymotrypsin and subtilisin Carlsberg as model systems. When the R-substituent in this series was changed from a p-chlorophenyl to a 1-naphthyl group, α-chymotrypsin, but not subtilisin, reversed its usual preference for l-enantiomers and bound more tightly to the d-enantiomer [Martichonok, V., & Jones, J. B. (1996) J. Am. Chem. Soc. 118, 950−958]. The structural factors responsible for the differences in stereoselectivity between the two enzymes have been explored by X-ray crystallographic examination of subtilisin Carlsberg and γ-chymotrypsin complexes of the l- and d-enantiomers of p-chlorophenyl and 1-naphthyl boronic acid derivatives. In both enzymes, the l-isomers of the inhibitors, which are more closely related to the natural l-amino acid substrates, form tetrahedral adducts, covalently linking the central boron atom and Oγ of the catalytic serine. The d-isomers, however, differ in the way they interact with subtilisin or γ-chymotrypsin. With subtilisin, both the d-p-chlorophenyl and d-1-naphthyl inhibitor complexes form covalent Ser Oγ-to-boron bonds, but with γ-chymotrypsin, the same inhibitors lead to novel tetrahedral adducts covalently linking both Ser195 Oγ and His57 Nε2 covalently via the boron atom.