Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process

Abstract

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.

Sorting and processing of secretory proteins

“…By expressing active furin together with various mutant furins, these authors confirm that activation occurs by an intramolecular autoproteolytic mechanism. Whether the furin pro-region is important for the proper folding of the enzyme, as is the case for subtilisin E [259][260][261][262], has not yet been determined. Further processing at as yet unidentified sites immediately Nterminal to the trans-membrane domain is also known to occur, leading to the generation of soluble (and released) 76-80 kDa forms [222,263].…”

Efficient secretion of attacin from insect fat‐body cells requires proper processing of the prosequence

“…The procarboxypeptidase B is enzymically inactive due to the prosequence [37] and part of the propeptide in subtilisin Carlsberg, from Bacillus licheniformis, is important for processing and secretion [38]. The prosequence may also protect intracellular membranes from exported toxins [39] or may be important for correct protein folding [40,411. The proteolysis at the two basic residues present in many prosequences often takes place in acidic vesicles in the regulated pathway.…”

A novel Plasmodium-specific prodomain fold regulates the malaria drug target SUB1 subtilase

“…Functions range from unspecific hydrolysis of substrates in the environment generating free amino acids as nutriments in bacteria, to precise maturation of inactive precursors into active polypeptides, such as the tightly regulated pro-hormone to hormone conversion in eukaryotes [1][2][3] . Subtilases are synthesized as an inactive precursor, the zymogen, which is characterized by the presence of a prodomain that plays a dual essential role of intramolecular chaperone and specific inhibitor of the cognate catalytic domain 4,5 . During secretion, the prodomain undergoes an autoprocessing maturation process before to be degraded, thus unlocking enzymatic activity.…”