title: The propeptide of subtilisin BPN' as a temporary inhibitor and effect of an amino acid replacement on its inhibitory activity.
abstract: The propeptide of subtilisin-family proteases is known to exhibit inhibitory activity toward a cognate protease in addition to its function as an intramolecular chaperone. For detailed investigation of its inhibitory properties, the propeptide of subtilisin BPN' was produced in Escherichia coli. Inhibitory activity measurements and electrophoresis showed that the propeptide was a temporary inhibitor, which was initially potent but was gradually degraded by subtilisin BPN' through specific intermediates. The main cleavage site was identified as Glu53-Lys54, with minor sites at Thr17-Met18 and Met21-Ser22, which were located in turn regions of the propeptide in the complex with subtilisin BPN'. Since the isolated propeptide has been shown not to form a tertiary structure, these results indicate that main digestions proceed through proteolytic attack of subtilisin toward the accessible sites of the propeptide in the complex with subtilisin. Therefore, replacement of Glu53 at the main cleavage site by Asp, which is a less favorable amino acid than Glu for subtilisin, makes the propeptide a more resistant temporary inhibitor.
authors.affiliation: Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kojima
authors.forename: T
authors.initials: T
authors.lastname: Minagawa
authors.forename: K
authors.initials: K
authors.lastname: Miura
mesh_terms: D001665:Binding Sites; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011480:Protease Inhibitors; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D013381:Subtilisins