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.
Mata
L
L
INRA, Unité de Recherche de Biochimie et Structure des Protéines, 78352 Jouy-en-Josas Cedex, France.
Gripon
J C
JC
Mistou
M Y
MY
D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D010447:Peptide Hydrolases; C028759:oligopeptidase; D000626:Aminopeptidases; C080203:PEPC proteinase; D012697:Serine Endopeptidases