title: A novel double-headed proteinaceous inhibitor for metalloproteinase and serine proteinase.
abstract: A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (Streptomyces caespitosus neutral proteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K(i) value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN' (K(i) = 1.4 nm) (EC ). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M(r) = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN' was identified to be Met(71)-Tyr(72) bond by specific cleavage. To identify the reactive site for ScNP, Tyr(33) and Tyr(72), which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN' but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN' was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN'.
authors.affiliation: Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Hiraga
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.forename: K
authors.initials: K
authors.lastname: Oda
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002401:Cathepsin B / Q000037:antagonists & inhibitors; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D010434:Pepsin A / Q000037:antagonists & inhibitors; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011549:Pseudomonas / Q000201:enzymology; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D013302:Streptomyces / Q000201:enzymology; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000737:chemistry; D013820:Thermolysin / Q000037:antagonists & inhibitors; D014361:Trypsin Inhibitors / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011506:Proteins; D015842:Serine Proteinase Inhibitors; C413210:Streptomyces caespitosus neutral proteinase inhibitor; D014361:Trypsin Inhibitors; D013381:Subtilisins; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D002401:Cathepsin B; D010434:Pepsin A; D008666:Metalloendopeptidases; C105831:almelysin; C099068:vimelysin; C106507:zinc endoprotease, Streptomyces caespitosus; D013820:Thermolysin