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 b acteria. Its structural and enzymatic properties closely resembles those of the bleomycin hydrolases, a group of cytoplasmic enzymes isolated from euk aryotes. Previous biochemical and structural data have shown that the C-ter minal end of PepC partially occupies the active site cleft. In this work th e substrate specificity of PepC was engineered by deletion of the four C-te rminal residues. The mutant PepC Delta 432-435 cleaved peptide substrates a s an oligopeptidase while the aminopeptidase specificity was totally abolis hed. The substrate size dependency indicated that PepC Delta 432-435 posses ses an extended binding site able to accommodate four residues of the subst rate on both sides of the cleaved bond. The activity of PepC Delta 432-435 towards tryptic fragments of casein revealed a preference for peptides with hydrophobic amino acids at positions P2 and P3 and for Gly, Asn and Gin at position P1. PepC Delta 432-435 was shown to be highly sensitive to the th iol 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 endopept idase from the papain family.