Maturation Processing and Characterization of Streptopain

• Published in: The Journal of biological chemistry 2003-05, Vol.278 (19), p.17336-17343
• Main Authors: Chen, Chiu-Yueh, Luo, Shih-Chi, Kuo, Chih-Feng, Lin, Yee-Shin, Wu, Jiunn-Jong, Lin, Ming T., Liu, Ching-Chuan, Jeng, Wen-Yih, Chuang, Woei-Jer
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Cysteine Endopeptidases - metabolism; Escherichia coli; Fibrinolysin; Molecular Sequence Data; Protein Folding; Protein Processing, Post-Translational; Recombinant Proteins - metabolism; Streptococcus pyogenes - enzymology; Trypsin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M209038200

Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination ofcis- and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn356 might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10- and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions.