Deletion of the four C-terminal residues of PepC converts an aminopeptidase into an oligopeptidase.

The aminopeptidase PepC is a cysteine peptidase isolated from lactic acid bacteria. Its structural and enzymatic properties closely resembles those of the bleomycin hydrolases, a group of cytoplasmic enzymes isolated from eukaryotes. Previous biochemical and structural data have shown that the C-terminal end of PepC partially occupies the active site cleft. In this work the substrate specificity of PepC was engineered by deletion of the four C-terminal residues. The mutant PepCDelta432-435 cleaved peptide substrates as an oligopeptidase while the aminopeptidase specificity was totally abolished. The substrate size dependency indicated that PepCDelta432-435 possesses an extended binding site able to accommodate four residues of the substrate on both sides of the cleaved bond. The activity of PepCDelta432-435 towards tryptic fragments of casein revealed a preference for peptides with hydrophobic amino acids at positions P2 and P3 and for Gly, Asn and Gln at position P1. PepCDelta432-435 was shown to be highly sensitive to the thiol peptidase inhibitors leupeptin or E64 which are inefficient towards the wild-type PepC. In conclusion, deletion of the four C-terminal residues in PepC produces a new enzyme with properties resembling those of an endopeptidase from the papain family.