In vitro processing of pro-subtilisin produced in Escherichia coli.
In a previous paper (Ikemura, H., Takagi, H., and Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864), we demonstrated that the pro-sequence consisting of 77 amino acid residues at the amino terminus of subtilisin is essential for the production of active subtilisin. When the aggregates of pro-subtilisin produced in Escherichia coli were solubilized in 6 M guanidine hydrochloride and dialyzed against 200 mM sodium phosphate buffer (pH 7.1 or 6.2), pro-subtilisin was efficiently processed to active subtilisin. When more than 14 residues were removed from the amino terminus of the pro-sequence, active subtilisin was no longer produced as in the in vivo experiments. Similarly, active subtilisin would not renature under the same conditions once solubilized in guanidine hydrochloride. When the aspartic acid residue at the active site (Asp32) was altered to asparagine, processing of mutant pro-subtilisin was not observed even in the presence of wild-type pro-subtilisin. Inhibitors such as phenylmethanesulfonyl fluoride or Streptomyces subtilisin inhibitor did not block the processing of wild-type pro-subtilisin. These facts indicate that processing or pro-subtilisin is carried out by an intramolecular, self-processing mechanism. When the sample was dialyzed against 20 mM sodium phosphate (pH 6.2), no active subtilisin was found, suggesting that the highly charged nature of the pro-sequence plays an important role in the process of refolding of denatured pro-subtilisin.
D001426:Bacterial Proteins / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009943:Organophosphorus Compounds / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D012995:Solubility; D013381:Subtilisins / Q000378:metabolism*
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; C018150:subtilisin inhibitor protein, Streptomyces; C057107:prosubtilisin; C008705:octamethyl pyrophosphoramide; D010664:Phenylmethylsulfonyl Fluoride; D013381:Subtilisins