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The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments. As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL). A series of mono-, di- and triacidic acid methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. Kinetic parameters for these chemically modified mutant (CMM) enzymes were determined at pH 8.6 under conditions which ensured complete ionization of the unnatural amino acid side-chains introduced. The presence of up to three negative charges in the S1, S1' and S2 subsites of SBL resulted in up to 11-fold lowered activity, possibly due to interference with oxyanion stabilization of the transition state of the hydrolytic reactions catalyzed. Each unit increase in negative charge resulted in a raising of K(M) and a reduction of k(cat). However, no upper limit was observed for increases in K(M), whereas decreases in k(cat) reached a limiting value. Comparison with sterically similar but uncharged CMMs revealed that electrostatic effects of negative charges at positions 62, 156 and 217 are detrimental, but are beneficial at position 166. These results indicate that the ground-state binding of SBL to the standard substrate, Suc-AAPF-pNA, to SBL is reduced, but without drastic attenuation of catalytic efficiency, and show that SBL tolerates high levels of charge at single sites.

Employing the strategy of combined site directed mutagenesis and chemical modification, we previously generated chemically modified mutant enzymes (CMMs) of subtilisin Bacillus lentus (SBL). We now report the use of these SBL-CMMs for peptide coupling reactions. The SBL-CMMs exhibit dramatically altered substrate specificity, including the acceptance of d -amino acid acyl donors, generating dipeptides containing d -Phe, d -Ala and d -Glu in up to 66% yield, which was not possible using wild-type SBL (WT-SBL). In addition, SBL-CMMs accommodate α-branched amino acids such as l -Ala-NH 2 as acyl acceptors in their S 1 ′ pockets, which WT-SBL will not.

A series of chemically modified mutants (CMMs) of subtilisin B. lentus (SBL) were generated employing the combination of site-directed mutagenesis and chemical modification. This strategy entails the mutation of a selected active site residue to cysteine and its subsequent modification with a methanethiosulfonate reagent CH3SO2S-R, where R may be infinitely variable. The present study was undertaken to evaluate the changes in specificity and pH-activity profiles that could be induced by modification of S156C and S166C in the S1 pocket of SBL with a representative range of side chain modifications, namely R −CH3, −CH2C6H5, −CH2CH2NH3+ and CH2CH2SO3−. The side chain of S156C is surface exposed and well solvated while that of S166C points into the pocket. Kinetic evaluation of the CMMs with suc-AAPF-pNA as substrate showed that the kcat/KMs changed very little for the S156C CMMs, but varied by up to 11-fold for the S166C CMMs. pH-Activity profiles were also determined, and showed that a negatively or positively charged side chain modification increased or decreased respectively, the pKa of the catalytic triad histidine for both modification sites but with more dramatic changes for the interior pointing S166C than for the solvent exposed S156C site. As an additional probe of altered specificity, inhibition of the CMMs by a representative series of 5 boronic acid transition state analogue inhibitors was determined. The KIs observed ranged from a 3.5-fold improvement over the WT value, to a 12-fold decrease in binding. Overall, greater variability in all the parameters measured, activity, pKa, and boronic acid binding resulted from modification at the inward pointing 166 site than at the solvent-exposed 156 site.

For synthetic applications of proteases, such as for peptide coupling, a combination of high esterase and low amidase activities is required. While achieving such specificity has been a long-standing goal, the decreases in amidase activity achieved to date have often also been accompanied by decreases in esterase activity. In the current study, a strategy of combined site-directed mutagenesis and chemical modification was applied to the serine protease subtilisin Bacillus lentus (SBL) to improve its esterase-over-amidase specificity. Using the crystal structure of SBL as a guide, the N62, L217, S166, and M222 active site residues were chosen for mutagenesis to cysteine and subsequent modification by alkyl methanethiosulfonate reagents. An initial rapid, combinatorial screen of the chemically modified mutant enzymes (CMMs) generated and, of their parent cysteine mutants, identified 25 promising candidates which were then subjected to detailed kinetic evaluations. Of these CMM and mutant enzymes, 20 exhibit...

Developing the ability to vary the pH−activity profile of an enzyme in a controlled manner has been a long sought-after goal. Such tailoring provides important insights into mechanism and permits optimization of enzyme performance in organic synthesis applications. The most successful approaches to date toward altering pH−activity profiles of enzymes have employed either site-directed mutagenesis or chemical modification to alter enzyme surface charge. We now report that, by combining these two methodologies, dramatic pKa changes can be induced in the serine protease subtilisin B. lentus. In particular, site specific incorporation of unnatural amino acid side chains by the following strategy, WT → Asn62Cysmutant + H3C−SO2−S−R → Asn62Cys-S−R, where R may be infinitely variable, has demonstrated that pKa shifts of up to 0.72 unit are achievable and are accompanied by significant activity enhancements. The most dramatic pKa shifts are caused by chemical modification with hydrophobic aliphatic moieties. A lin...

Specificity differences between the S1-pockets of subtilisin B. lentus (SBL), and its M222C/S mutants have been explored with boronic acid inhibitors. Similar binding trends were noted, with 2,4-dichlorophenylboronic acid being the best overall inhibitor for each enzyme. In addition, a correlation between inhibitor binding and the electrophilicity of boron was found for both the M222C and M222S enzymes. Specificity differences between the S 1-pockets of subtilisin from B. lentus (SBL), and its M222C/S mutants, have been explored with boronic acid inhibitors. Similar binding trends were noted, with 2,4-dichlorophenyl boronic acid being the best overall inhibitor for each enzyme. In addition, a correlation between inhibitor binding and the electrophilicity of boron was found for both M222C and M222S enzymes.

A transition state analogue inhibitor, boronic acid benzophenone (BBP) photoprobe, was used to study the differences in the topology of the S1 pocket of chemically modified mutant enzymes (CMMs). The BBP proved to be an effective competitive inhibitor and a revealing active site directed photoprobe of the CMMs of the serine protease subtilisin Bacillus lentus (SBL) which were chemically modified with the hydrophobic, negatively charged and positively charged moieties at the S1 pocket S166C residue. As expected, in all cases BBP bound best to WT-SBL. BBP binding to S166C-SCH2C6H5 and S166C-CH2-c-C6H11, with their large hydrophobic side chains, was reduced by 86-fold and 9-fold, respectively, compared to WT. Relative to WT, BBP binding to the charged CMMs, S166C-S-CH2CH2SO3− or S166C-S-CH2CH2NH3+, was reduced 170-fold and 4-fold respectively. Photolysis of the WT-SBL–BBP enzyme–inhibitor (EI) complex, inactivated the enzyme and effected the formation of a covalent crosslink between WT and BBP. The crosslink was identified at Gly127 by peptide mapping analysis and Edman sequencing. Gly127 is located in the S1 hydrophobic pocket of SBL and its modification thus established binding of the benzophenone moiety in S1. Photolysis of the EI complex of S166C-SCH2C6H5, S166C-S-CH2CH2SO3−, or S166C-S-CH2CH2NH3+ and BBP under the same conditions did not inactivate these enzymes, nor effect the formation of a crosslink. These results corroborated the kinetic evidence that the active site topology of these CMMs is dramatically altered from that of WT. In contrast, while photolysis of the S166C-CH2-c-C6H11–BBP EI complex only inactivated 50% of the enzyme after 12 h, it still effected the formation of a covalent crosslink between the CMM and BBP, again at Gly127. However, this photolytic reaction was less efficient than with WT, demonstrating that the S1 pocket of S166C-CH2-c-C6H11 is significantly restricted compared to WT, but not as completely as for the other CMMs.

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 -CH(3) 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-CH(2)-c-C(6)H(11) 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 P(1) 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 k(cat)/K(M) 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-CH(2)CH(2)C(COO(-))(3) CMM. Conversely, the positively charged S166C-S-CH(2)CH(2)NH(3)(+) CMM generated showed a 19-fold improvement in k(cat)/K(M) for the suc-AAPE-pNA substrate and a 54-fold improvement in suc-AAPE-pNA/suc-AAPF-pNA selectivity relative to WT-SBL.

By combining site-directed mutagenesis with chemical modification, we have altered the S1 and S1' pocket specificity of subtilisin Bacillus lentus (SBL) through the incorporation of unnatural amino acid moieties, in the following manner: WT --Cysmutant + 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 S1' pocket of SBL, caused dramatic changes in kcat/KM, of an up to 122-fold decrease, while modification of S166C, which is located at the bottom of the S1 pocket and is partially surface exposed, effected more modest activity changes. Introduction of a positive charge at S166C does not alter kcat/KM, 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 S1 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 P1 residue of the AAPF product inhibitor.

Chemical Modification of Cysteine Mutants of Subtilisin Bacillus lentus Can Create Better Catalysts Than the Wild-Type Enzyme