Site-directed mutagenesis combined with chemical modification as a strategy for altering the specificity of the S1 and S1′ pockets of subtilisin Bacillus lentus

By combining site-directed mutagenesis with chemical modification, we have altered the S-1 and S-1' pocket specificity of subtilisin Bacillus lentus (SBL) through the incorporation of unnatural amino acid moieties, in the following manner: WT --> Cys(mutant) + H3CSO2SR --> Cys-SR, where R may be infinitely variable. A paradigm between extent of activity changes and surface exposure of the modified residue has emerged. Modification of M222C, a buried residue in the S-1' pocket of SBL, caused dramatic changes in k(cat)/K-M, of an up to 122-fold decrease, while modification of S166C, which is located at the bottom of the S-1 pocket and is partially surface exposed, effected more modest activity changes. Introduction of a positive charge at S166C does not alter k(cat)/K-M, whereas the introduction of a negative charge results in lowered activity, possibly due to electrostatic interference with oxyanion stabilization. Activity is virtually unaltered upon modification of S156C, which is located toward the bottom of the S-1 pocket and surface exposed and whose side chain is solvated. An unexpected structure-activity relationship was revealed for S166C-SR enzymes in that the pattern of activity changes observed with increasing steric size of R was not monotonic. Molecular modeling analysis was used to analyze this unprecedented structure-activity relationship and revealed that the position of the beta-carbon of Cys166 modulates binding of the P-1 residue of the AAPF product inhibitor.