| Research Abstract |
In order to design new inhibitors for enzymes, we have investigated the structure-function relationship of the well-characterized protease inhibitor, Streptomyces subtilisin inhibitor (SSI), by the method of gene engineering technique. First, replacement of the amino acid at P1 site, the center of reactive site, of SSI by Lys or Arg resulted in attainment of inhibitory activity for trypsin, and by Tyr, Phe, or Trp resulted in that for chymotrypsin. These mutants inhibited subtilisin as strongly as wild type, whereas inhibitory activity of the mutants whose P1 site residue was Glu, Ile, or Pro was decreased. These observations demonstrated that inhibitory activities of P1 site mutants of protease inhibitors are related to the substrate specificity of protease. Substitution of the P4 site residue (Met70) of the mutant SSI with Lys at P1 site by Gly, and the mutant SSI with Gly at P1 site by Phe resulted in increase of inhibitory activity toward trypsin and subtilisin, respectively. These results showed that stronger inhibitors could be produced by replacements of amino acid residues around the reactive site of inhibitors, so that they were complementary to the subsite structure of protease. Furthermore, removal of disulfide dridge near the reactive site of SSI by replacements of Cys to Ser resulted in conversion of SSI into temporary inhibitor, in which inhibitory activity for the protease decreased with increasing incubation time after mixing with subtilisin. This finding suggested that conformational rigidity around the reactive site was required for the inhibitory action of SSI.
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