The autocatalytic processing of the subtilisin Carlsberg pro-region is independent of the primary structure of the cleavage site.
Subtilisins are extracellular seryl-proteases produced by bacilli (Markland and Emil, 1971). In addition to signal sequences, these proteases have N-terminal extensions (pro-regions) which have also been identified in several other proteases (Silen et al., 1988; Vasantha et al., 1984; Polhner et al., 1987; Henderson et al., 1987; Yanagida et al., 1986; Takagi et al., 1985). The pro-region holds the pro-protease associated with the membrane and release of the protease takes place as a result of pro-region removal by autocatalytic processing (Egnell and Flock, 1991). In this report we describe the construction of four deletion-mutations in the gene encoding subtilisin Carlsberg at the junction between the pro-region and mature subtilisin Carlsberg. We found that the introduction of different deletions abolished the ability of subtilisin to undergo autocatalytic cleavage of the pro-region in cis, whereas cleavage by exogenous subtilisin could still occur in trans. Point mutations were also introduced in positions -5 to +4 around the pro-region and native subtilisin cleavage site. Processing of pro-subtilisin with the point mutations showed that the autocatalytic cleavage and recognition of this junction of the subtilisin Carlsberg pro-region is independent of the amino acid sequence around the cleavage site.
Egnell
P
P
Centre for Biotechnology, Karolinska Institute, Novum, Huddinge, Sweden.
Flock
J I
JI
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*
D004792:Enzyme Precursors; D010446:Peptide Fragments; C057107:prosubtilisin; D013381:Subtilisins