Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin

We have previously shown that replacing the Pi-site residue (Ala) of chicke n ovomucoid domain 3 (OMCHI3) with a Met or Lys results in the acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively. Howeve r, the inhibitory activities thus induced are not strong. In the present st udy, we introduced additional amino acid replacements around the reactive s ite to try to make the P1-site mutants more effective inhibitors of chymotr ypsin or trypsin. The amino acid replacement Asp-->Tyr at the P2' site of O MCHI3(P1Met) resulted in conversion to a 35000-fold more effective inhibito r of chymotrypsin with an inhibitor constant (Ki) of 1.17x10(-11) M, The Ki value of OMCHI3(P1Met, P2'Ala) indicated that the effect on the interactio n with chymotrypsin of removing a negative charge from the P2' site was gre ater than that of introducing an aromatic ring. Similarly, enhanced inhibit ion of trypsin was observed when the Asp-->Tyr replacement was introduced i nto the P2' site of OMCHI3(P1Lys). Two additional replacements, Asp-->Ala a t the P4 site and Arg-->Ala at the P3' site, made the mutant a more effecti ve inhibitor of trypsin with a K-i value of 1.44x10(-9) M, By contrast, Arg -->Ala replacement at the P3' site of OMCHI3(P1Met, P2'Tyr) resulted in a g reatly reduced inhibition of chymotrypsin, and Asp-->Ala replacement at the P4 site produced only a small change when compared with a natural variant of OMCHI3. These results clearly indicate that not only the PI-site residue but also the characteristics, particularly the electrostatic properties, o f the amino acid residues around the reactive site of the protease inhibito r determine the strength of its interactions with proteases, Furthermore, a mino acids with different characteristics are required around the reactive site for strong inhibition of chymotrypsin and trypsin.