Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus:functional and structural consequences of mutation at the S4 substrate binding pocket

Serine endoproteases such as trypsins and subtilisins are known to have an extended substrate binding region that interacts with residues P6 to P3' of a substrate. In order to investigate the structural and functional effects of replacing residues at the S4 substrate binding pocket, the serine protease from the alkalophilic Bacillus strain PB92, which shows homology with the subtilisins, was mutated at positions 102 and 126-128. Substitution of Val102 by Trp results in a 12-fold increase in activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpNA). An X-ray structure analysis of the V102W mutant shows that the Trp side chain occupies a hydrophobic pocket at the surface of the molecule leaving a narrow crevice for the P4 residue of a substrate. Better binding of sAAPFpNA by the mutant compared with the wild type protein as indicated by the kinetic data might be due to the hydrophobic interaction of Ala P4 of the substrate with the introduced Trp102 side chain. The observed difference in binding of sAAPFpNA by protease PB92 and thermitase, both of which possess a Trp at position 102, is probably related to the amino acid substitutions at positions 105 and 126 (in the protease PB92 numbering). Kinetic data for the variants obtained by random mutation of residues Ser126, Pro127 and Ser128 reveal that the activity towards sAAPFpNA increases when a hydrophobic residue is introduced at position 126. An X-ray diffraction analysis was carried out for the three protease PB92 mutants which have residues Ser126-Pro127-Ser128 replaced by Met-Ala-Gly ('MAG' mutant), Phe-Gln-Ser ('FQS' mutant) and Asn-Ser-Ala ('NSA' mutant). Met126 and Phe126 in the crystal structures of the corresponding mutants are fixed in the same hydrophobic environment as Trp102 in the V102W mutant. In contrast, Asn126 in the 'NSA' mutant is completely disordered in both crystal forms for which the structure has been determined. According to our kinetic measurements none of the mutants with Met, Phe, Leu or Val at position 126 binds sAAPFpNA better than the wild type enzyme. Results of the site-directed mutagenesis at position 127 imply that possible interaction of this residue with a substrate has almost no effect on activity towards sAAPFpNA and casein.