Toward Tailoring the Specificity of the S₁ Pocket of Subtilisin B. lentus:  Chemical Modification of Mutant Enzymes as a Strategy for Removing Specificity Limitations

Abstract

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.

Enzyme Redesign

“…Site-directed mutagenesis has been successful in redesigning the substrate specificity of a large number of common classes of enzymes, including oxidoreductases (dehydrogenases − and trypanothione/glutathione reductases − ), hydrolases (acetylcholinesterases, − β-lactamases, − and proteases − ), transferases (aminotransferases − and glutathione- S -transferase , ), and restriction enzymes. − These experiments demonstrate that rational approaches based on a three-dimensional structure or amino acid sequence alignments can succeed at changing ligand recognition. In each of the examples that follow the chemical mechanism of the reaction remains unaltered.…”

Generation of New Enzymes via Covalent Modification of Existing Proteins

“…The presence of up to three negative charges in the various SBL subsites resulted in up to 11-fold decreased activity using a normal SBL substrate, suc-AAPF-pNA. The mutant at position 166 was chosen for additional studies which examined the effects of anionic chemical substitutions ( 8 − 8 through 8 − 10 ) on the hydrolysis of a charge-complementary cationic substrate . The resulting constructs showed up to 9-fold improvements in k cat / K M for the substrate su-AAPR-pNA and a concomitant 61-fold improvement in suc-AAPR-pNA/suc-AAPF-pNA selectivity.…”

Enhancement of the Thermostability of a Fibrinolytic Enzyme from Bacillus amyloliquefaciens CH51

“…Earlier mutagenesis study of B. subtilis subtilisin E indicated that the residues of Gly-166 and Asn-218 are involved in substrate binding site, which explains their effects on catalytic activity and thermostability [6,27]. Gly-166 and Asn-218 were targeted to change the amino acid residues in AprE51.…”