Toward tailoring the specificity of the S1 pocket of subtilisin B-lentus:: Chemical modification of mutant enzymes as a strategy for removing specificity limitations

In both protein chemistry studies and organic synthesis applications, it is desirable to have available a toolbox of inexpensive proteases with high selectivity and diverse substrate preferences. Toward this goal, we have generated a series of chemically modified mutant enzymes (CMMs) of subtilisin B. lentus (SBL) possessing expanded S-1 pocket specificity. Wild-type SBL shows a marked preference for substrates with large hydrophobic P-1 residues, such as the large Phe P-1 residue of the standard suc-AAPF-pNA substrate. To confer more universal P-1 specificity on S-1, a strategy of chemical modification in combination with site-directed mutagenesis was applied. For example, WT-SBL does not readily accept small uncharged P-1 residues such as the -CH3 side chain of alanine. Accordingly, with a view to creating a S-1 pocket that would be of reduced volume providing a better fit for small P-1 side chains, a large cyclohexyl group was introduced by the CMM approach at position S166C with the aim of partially filling up the S-1 pocket. The S166C-S-CH2-c-C6H11 CMM thus created showed a 2-fold improvement in k(cat)/K-M with the suc-AAPA-pNA substrate and a 51-fold improvement in suc-AAPA-pNA/suc-AAPF-PNA selectivity relative to WT-SBL. Furthermore, WT-SBL does not readily accept positively or negatively charged pi residues. Therefore, to improve SBL's specificity toward positively and negatively charged P-1 residues, we applied the CMM methodology to introduce complementary negatively and positively charged groups, respectively, at position S166C in S-1. A series of mono-, di-, and trinegatively charged CMMs were generated and all showed improved k(cat)/K(M)s With the positively charged P-1 residue containing substrate, suc-AAPR-pNA. Furthermore, virtually arithmetic improvements in k(cat)/K-M were exhibited with increasing number of negative charges on the S166C-R side chain. These increases culminated in a 9-fold improvement in kcat/KM for the suc-AAPR-pNA substrate and a 61-fold improvement in suc-AAPR-pNA/suc-AAPF-PNA selectivity compared to WT-SBL for the trinegatively charged S166C-S-CH2CH2C(COO-)(3) CMM. Conversely, the positively charged S166C-S-CH2CH2NH3+ CMM generated showed a 19-fold improvement in kcat/KM for the suc-AAPE-pNA substrate and a 54-fold improvement in suc-AAPE-pNA/suc-AAPF-pNA selectivity relative to WT-SBL.