Protein Engineering, Vol. 12, No. 8, 681-686,
August 1999
© 1999 Oxford University Press
Deletion of the four C-terminal residues of PepC converts an aminopeptidase into an oligopeptidase
INRA, Unité de Recherche de Biochimie et Structure des Protéines, 78352 Jouy-en-Josas Cedex, France
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 PepC
432–435 cleaved peptide substrates as an oligopeptidase while the aminopeptidase specificity was totally abolished. The substrate size dependency indicated that PepC432–435 possesses an extended binding site able to accommodate four residues of the substrate on both sides of the cleaved bond. The activity of PepC432–435 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. PepC432–435 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.
Keywords: bleomycin hydrolase/cystein proteinaseLactococcus/papain/substrate specificity