The role of tryptophan residues in the autoprocessing of prosubtilisin E.
Subtilisin E, a serine protease from Bacillus subtilis, requires an N-terminal propeptide for its correct folding. The propeptide is autocleaved and digested by the subtilisin domain upon proper folding. Here we investigated the individual roles of the three Trp residues within the subtilisin domain (Trp106, Trp113 and Trp241) on propeptide processing, enzymatic activity and stability of subtilisin. When the propeptide processing was examined by SDS-PAGE after refolding by rapid dilution, the mutation at either position Trp106 or Trp113 was found to significantly delay the propeptide processing, while the mutation at Trp241 had no effect. Far-UV circular dichroism (CD) spectra of the mutants revealed that the mutations at the three positions did not affect appreciably the alpha-helix content of subtilisin. Secondary structure thermal unfolding monitored by CD spectroscopy revealed that none of the tryptophan residues had any significant effect on the stability of mature subtilisin. The enzymatic activity measurements showed that only Trp106 plays a major role in the enzymatic activity of subtilisin E. These results demonstrate that both Trp106 and Trp113 play a specific role in propeptide processing and enzymatic activity, while Trp241 plays no considerable role on any of these activities.
Sone
Michio
M
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Falzon
Liliana
L
Inouye
Masayori
M
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D010446:Peptide Fragments; C057107:prosubtilisin; D014364:Tryptophan; D013381:Subtilisins