Plasminostreptin, a protein proteinase inhibitor produced by Streptomyces antifibrinolyticus. II. Determination of the reactive site for proteinases.

Chemical modification of the amino groups of plasminostreptin with 2,4,6-trinitrobenzene sulfonic acid or O-methylisourea completely abolished its inhibitory activities against trypsin (EC 3.4.21.4) and plasmin (EC 3.4.21.7). This indicated that plasminostreptin requires at least 1 lysine residue for its trypsin and plasmin inhibitory activities. Incubation of plasminostreptin with a small amount of trypsin at pH 3.5 resulted in the conversion of the virgin plasminostreptin into a modified form. Reduction and Scarboxymethylation of this trypsin-modified plasminostreptin led to the formation of two fragments, the sum of the amino acid compositions of which corresponded to the amino acid composition of the virgin plasminostreptin. The larger fragment, composed of 69 amino acid residues, including 3 S-carboxymethylcysteine residues, had the original NH,-terminal glycine and the newly formed COOHterminal lysine. The smaller fragment, composed of 40 amino acids, including 1 S-carboxymethylcysteine residue, had the newly formed NH,-terminal glutamine and the original COOH-terminal phenylalanine. Thus, it became clear that upon hydrolysis with trypsin, only the Lys+Gln’O bond is selectively cleaved and that the two fragments are held together by a disulfide bridge. Subtilisin-modified plasminostreptin, produced by incubation with a small amount of subtilisin (EC 3.4.21.14) at pH 4.0, had a newly generated NH,-terminal sequence, GlnPhe-Asp, in addition to the original sequence, Gly-Leu-Tyr, and a newly generated COOH-terminal lysine in addition to the original phenylalanine. These results show that the Lys-Gln bond is specifically split by limited proteolysis with trypsin or subtilisin, and it was concluded that the Ly8s-Gln70 bond contained within a disulfide bridge in the plasminostreptin molecule is the reactive site for the inhibition of trypsin and subtilisin. A plasminostreptin derivative which was deprived of 6 amino acid residues in the COOH-terminal region did not retain proteinase inhibitory activity at all. Therefore, beside the reactive site, the presence of at least 6 amino acid residues