title: Evidence for intramolecular processing of prosubtilisin sequestered on a solid support.
abstract: Subtilisin E is synthesized in Bacillus subtilis as a preprosubtilisin. The prepeptide is removed by a signal peptidase, and the propeptide is cleaved from the mature protein by the catalytic domain of subtilisin itself in an autocatalytic fashion. A six residue histidine-tag was attached to the C terminus of prosubtilisin and mature subtilisin to enable immobilization on a metal chelating resin. Guanidine-HC1 denatured histidine-tagged subtilisin and prosubtilisin were immobilized on Co2+ charged Talon resin, then renatured by dialysis of the resin against renaturation buffer. Refolding of the immobilized prosubtilisin resulted in its quantitative autoprocessing and the formation of active enzyme. Mature subtilisin on the other hand refolded into an active conformation with very low efficiency, and at the same concentration the steady-state rate attained was at least a 1000 times lower than that from prosubtilisin. The results give very strong support for an intramolecular autoprocessing pathway for prosubtilisin, in addition to an intermolecular one demonstrated before. The results also demonstrate rather convincingly the very much higher yield of active enzyme refolded from prosubtilisin than from mature protein under sequestered unimolecular conditions.
authors.affiliation: Department of Chemistry, Rutgers, State University of New Jersey, Newark 07102, USA.
authors.forename: A
authors.initials: A
authors.lastname: Volkov
authors.forename: F
authors.initials: F
authors.lastname: Jordan
mesh_terms: D004792:Enzyme Precursors / Q000378:metabolism*; D004800:Enzymes, Immobilized; D006868:Hydrolysis; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D013381:Subtilisins / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011994:Recombinant Proteins; C057107:prosubtilisin; D013381:Subtilisins