Autoprocessing of prothiolsubtilisin E in which active-site serine 221 is altered to cysteine.
Subtilisin, an extracellular serine protease from Bacillus subtilis, requires the amino-terminal propeptide of 77 amino acid residues for the formation of the active enzyme. The propeptide is cleaved upon completion of folding. Serine 221 at the active center was substituted with cysteine, and the mutant enzyme (prothiolsubtilisin) was expressed in Escherichia coli under the control of a T7 promoter. Prothiosubtilisin, which was produced as inclusion bodies, was dissolved in 6 M guanidine HCl and purified to near homogeneity in the presence of 5 M urea. The purified protein was renatured by stepwise dialysis. In spite of the mutation at the active center, the propeptide was found to be autoprocessed with approximately 60-80% efficiency. However, protease activity could not be detected in the final product by the spectrophotometric assay. Moreover, the cleaved propeptide remained tightly bound to thiolsubtilisin without being digested, as evident by SDS-polyacrylamide gel electrophoresis. The amino-terminal sequence of the processed thiolsubtilisin was determined and proved that the propeptide was cleaved at a site identical to that of wild-type prosubtilisin. The processed thiolsubtilisin was also found to contain one free SH group/molecule. These results unambiguously demonstrate that the processing of prosubtilisin occurs by an intramolecular autoprocessing mechanism.
Li
Y
Y
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Inouye
M
M
D001412:Bacillus subtilis / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012694:Serine; D013381:Subtilisins; D003545:Cysteine