Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes.
A recombinant streptococcal C5a peptidase was expressed in Escherichia coli and its catalytic properties and thermal stability were subjected to examination. It was shown that the NH2-terminal region of C5a peptidase (Asn32-Asp79/Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease streptopain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range of temperatures between 40 and 43 degrees C. The process of heat denaturation of C5a peptidase investigated by fluorescence and circular dichroism spectroscopy revealed that the protein undergoes biphasic unfolding transition with Tm of 50 and 70 degrees C suggesting melting of different parts of the molecule with different stability. Unfolding of the less stable structures was accompanied by the loss of proteolytic activity. Using synthetic peptides corresponding to the COOH-terminus of human complement C5a we demonstrated that in vitro peptidase catalyzes hydrolysis of two His67-Lys68 and Ala58-Ser59 peptide bonds. The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis of its derivative Ac-SQLRANISH-pNA that lacks residues at P2'-P7' positions, suggest the importance of C5a peptidase interactions with the P' side of the substrate.
D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D018829:Adhesins, Bacterial; D004269:DNA, Bacterial; D004792:Enzyme Precursors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010450:Endopeptidases; C053088:C5a peptidase