Maturation processing and characterization of streptopain.
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 of cis- 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 Asn(356) 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.
Chen
Chiu-Yueh
CY
Department of Biochemistry, National Cheng Kung University College of Medicine, 1 University Road, Tainan 701, Taiwan.
Luo
Shih-Chi
SC
Kuo
Chih-Feng
CF
Lin
Yee-Shin
YS
Wu
Jiunn-Jong
JJ
Lin
Ming T
MT
Liu
Ching-Chuan
CC
Jeng
Wen-Yih
WY
Chuang
Woei-Jer
WJ
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli; D005341:Fibrinolysin; D008969:Molecular Sequence Data; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000201:enzymology*; D014357:Trypsin
D011994:Recombinant Proteins; D014357:Trypsin; D005341:Fibrinolysin; D003546:Cysteine Endopeptidases; C042477:streptopain