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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

Luis Mata, Jean-Claude Gripon and Michel-Yves Mistou1

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{Delta}432–435 cleaved peptide substrates as an oligopeptidase while the aminopeptidase specificity was totally abolished. The substrate size dependency indicated that PepC{Delta}432–435 possesses an extended binding site able to accommodate four residues of the substrate 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 Gln at position P1. PepC{Delta}432–435 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

1 To whom correspondence should be addressed


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