Restriction of substrate specificity of subtilisin E by introduction of a side chain into a conserved glycine residue.

Substitution of the conserved Gly127 for residues having a side chain markedly changed the substrate specificity of subtilisin E from Bacillus subtilis. The crystallographic findings suggested that Gly127 is responsible for accepting even the large P1 substrates, and the marked change of specificity was attributed to the introduction of a side chain in this position. To test this hypothesis, Gly127 was replaced with 3 non-charged amino acids, Ala, Ser and Val. When assayed with synthetic peptide substrates, all mutants purified from the periplasmic space in Escherichia coli showed a marked preference for small P1 substrate up to 150-fold relative to the wild-type. The kinetic data and molecular modeling analysis suggest that large hydrophobic P1 residues were unable to access the binding pocket due to steric hindrance.