CHYMOTRYPSIN INHIBITOR FROM ERYTHRINA-VARIEGATA SEEDS - INVOLVEMENT OF AMINO-ACID-RESIDUES WITHIN THE PRIMARY BINDING LOOP IN POTENT INHIBITORY ACTIVITY TOWARD CHYMOTRYPSIN

The stoichiometry of the interaction between Erythrina variegata chymo trypsin inhibitor ECI and chymotrypsin was reinvestigated by analysis of their complex with ultracentrifugation and with amino acid analysis of the components separated. The amino acid analysis clearly showed t hat the stoichiometry of ECI and chymotrypsin was 1:1, though the appa rent molecular mass of the complex was estimated to be 60 kDa. To exam ine the contribution of Leu64 (the P1 residue) to the inhibitory activ ity of ECI, a complete set of mutated inhibitors in which the amino ac id at position 64 was replaced by 19 other amino acid residues was con structed by means of site-directed mutagenesis. Potent inhibitory acti vities (Ki, 1.3-4.6x10(-8) M) exceeding that of the wild-type ECI (K-i , 9.8x10(-8) M) were present in the mutant proteins L64F, L64M, L64W, and L64Y. The inhibitory activity of the mutant L64R was practically i dentical to that of the wild-type ECI. All other mutants exhibited sli ghtly decreased inhibitory activities with K-i values of 1.9-4.6x10(-7 ) M. These results indicate that ECI-chymotrypsin interaction involves not only the P1 site residue but also other residue(s) of ECI. A seri es of individual alanine mutations was then constructed in residues Gl n62 (P3), Phe63 (P2), Ser65 (P1'), Thr66 (P2'), and Phe67 (P3') in ord er to evaluate the contribution of each residue in the primary binding loop to the inhibitory activity. Replacement of Gln62, Phe63, and Phe 67 with Ala residues decreased the inhibitory activity, the K-i values being increased by approximately 3-4-fold; but replacement of Ser65 a nd Three had relatively little effect. This suggests that the P2, P3, and P3' residues, together with the P1 residue, in the primary binding loop play an important role in the inhibitory activity toward chymotr ypsin.