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Pro‐subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro
Author(s) -
Ohta Y.,
Inouye M.
Publication year - 1990
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1990.tb00596.x
Subject(s) - subtilisin , urea , biochemistry , active site , biology , size exclusion chromatography , guanidine , chromatography , chemistry , enzyme
Summary The formation of active subtilisin E from pro‐subtilisin E requires the removal of the N ‐terminal pro‐sequence of 77 residues. Pro‐subtilisin E produced in Escherichia coli using a pINIII‐ompA vector was first extracted with 6M guanidine‐HCl and 5M urea and purified to homogeneity in the presence of 5M urea. Upon drop dialysis against 0.2M sodium phosphate buffer (pH 6.2), the purified pro‐subtilisin in 5M urea was processed to active subtilisin of which the N ‐terminal sequence and migration in SDS‐polyacrylamide gel electrophoresis were identical to those of authentic active subtilisin E. This process was found to be very sensitive to the ionic strengths and anions used. Under the optimum conditions (dialysis against 0.5 M (NH 4 ) 2 SO 4 and 1 mM CaCl 2 in 10mM Tris‐HCl buffer (pH 7.0) at 4°C for 1 h), approximately 20% of pro‐subtitisin E was converted to active subtilisin E. The activation process was not inhibited by Streptomyces subtilisin inhibitor, and pro‐subtiiisin E in which the active site was mutated (Asp 32 to Asn) was unable to be processed under the optimum conditions. These results confirmed the previous hypothesis that the processing of pro‐subtilisin occurs by an intramolecular, autoprocessing mechanism.