Autocatalytic processing of the streptococcal cysteine protease zymogen - Processing mechanism and characterization of the autoproteolytic cleavage sites

The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds throug h the sequential appearance of at least six intermediates, five of which we re characterized through a combination of N-terminal sequencing and MS. Int ermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time-course studies of the proSCP processing ga ve a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A simil ar sequential appearance of intermediates was observed when inactive Cys192 Ser proSCP was treated with native, enzymatically active SCP, thus demonstr ating that the maturation can exclusively proceed by a bimolecular mechanis m. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whe ther the amino acid sequences at the processing sites could be used for dev eloping new, specific substrates, 3-amino benzoic acid octapeptide derivati ves based on all five characterized amino acid sequences from the autoproce ssing cleavage sites were synthesized and tested for activity. The 3-amino benzoic acid derivatives have k(cat)/K-M values ranging from 1200 to 7700.M -1.s(-1), making them very good endopeptidase substrates for SCP.