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Evidence for intramolecular processing of prosubtilisin sequestered on a solid support

A Volkov1, F Jordan

  • 1Department of Chemistry, Rutgers, State University of New Jersey, Newark 07102, USA.

Journal of Molecular Biology
|October 11, 1996
PubMed
Summary

Prosubtilisin refolding yields active subtilisin E enzyme efficiently via autoprocessing. Mature subtilisin refolding is inefficient, producing significantly less active enzyme, supporting intramolecular processing.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Subtilisin E from Bacillus subtilis is initially synthesized as a preprosubtilisin.
  • Processing involves signal peptidase removal of the prepeptide and autocatalytic cleavage of the propeptide by subtilisin's catalytic domain.

Purpose of the Study:

  • To investigate the refolding efficiency and activity of immobilized prosubtilisin versus mature subtilisin.
  • To explore the intramolecular autoprocessing pathway of prosubtilisin.

Main Methods:

  • Histidine-tagging of prosubtilisin and mature subtilisin for immobilization on Co2+ charged Talon resin.
  • Denaturation with Guanidine-HCl followed by renaturation via dialysis.
  • Assessing enzyme activity and autoprocessing efficiency post-refolding.

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Main Results:

  • Immobilized prosubtilisin quantitatively autoprocessed to form active subtilisin E upon refolding.
  • Mature subtilisin refolded with very low efficiency, yielding an enzyme with at least 1000-fold lower steady-state rate compared to prosubtilisin.
  • Refolding prosubtilisin resulted in a significantly higher yield of active enzyme than refolding mature subtilisin under unimolecular conditions.

Conclusions:

  • The study strongly supports an intramolecular autoprocessing pathway for prosubtilisin, complementing previously demonstrated intermolecular pathways.
  • Prosubtilisin serves as a more efficient precursor for generating active subtilisin E compared to the mature enzyme, particularly under conditions favoring unimolecular refolding.