Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process.
Subtilisin E, an alkaline serine protease consisting of a single polypeptide chain of 275 amino acids is produced from a pre-pro-protein. The pre-sequence functions as the signal peptide for protein secretion across the membrane. Deletion of the pro-sequence yields mature but inactive subtilisin: the 77-amino acid pro-sequence must precede the mature subtilisin to guide the latter into an active conformation. Pro-subtilisin denatured in 6 M guanidine-HCl can be self-processed to the active enzyme intramolecularly, with concomitant cleavage of the pro-sequence, when dialysed against renaturing buffer. We have constructed an active-centre mutant of pro-subtilisin (Asp 32----Asn) which is not processed to active enzyme, unlike the wild-type pro-subtilisin, because intramolecular processing is prevented. Here we report an intermolecular pathway for the refolding of the inactive mature protein to an active enzyme in vitro with the aid of exogenously added pro-sequence. We establish conditions under which the mature inactive form, as well as acid-denatured subtilisins Carlsberg and BPN', can be renatured by the mutant pro-subtilisin.
Zhu
X L
XL
Department of Chemistry, Rutgers State University of New Jersey, Newark.
Ohta
Y
Y
Jordan
F
F
Inouye
M
M
D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism
D004792:Enzyme Precursors; D010446:Peptide Fragments; D011994:Recombinant Proteins; C057107:prosubtilisin; D013381:Subtilisins