Conformation of the primary binding loop folded through an intramolecular interaction contributes to the strong chymotrypsin inhibitory activity of the chymotrypsin inhibitor from Erythrina variegata seeds

We previously demonstrated that amino acid residues Gln62 (P3), Phe63 (P2), Leu64 (P1), and Phe67 (P3') in the primary binding loop of Erythrina varie gata chymotrypsin inhibitor (ECI), a member of the Kunitz inhibitor family, are involved in its strong inhibitory activity toward chymotrypsin [Iwanag a et al. (1998) J. Biochem. 124, 663-669]. To determine whether or not thes e four amino acid residues predominantly contribute to the strong inhibitor y activity of ECI, they were simultaneously replaced by Ala. The results sh owed that a quadruple mutant, Q62A/F63A/L64A/F67A, retained considerable in hibitory activity (K-i, 5.6 X 10(-7) M), indicating that in addition to the side chains of these four amino acid residues, the backbone structure of t he primary binding loop in ECI is essential for the inhibitory activity tow ard chymotrypsin. Two chimeric proteins, in which the primary binding loops of ECI and ETIa were exchanged: an isoinhibitor from E. variegata with low er chymotrypsin inhibitory activity, were constructed to determine whether the backbone structure of the primary binding loop of ECI was formed by the amino acid residues therein, or through an interaction between the primary binding loop and the residual structure designated as the "scaffold." A ch imeric protein, ECI/ETIa, composed of the primary binding loop of ECI and t he scaffold of ETIa showed weaker inhibitory activity (K-i, 1.3 X 10(-6) M) than ECI (K-i, 9.8 X 10(-8) M). In contrast, a chimera, ETIa/ECI, comprisi ng the primary binding loop of ETIa and the scaffold of ECI inhibited chymo trypsin more strongly (K-i, 5.7 X 10(-7) M) than ETIa (K-i, 1.3 X 10(-6) M) . These results indicate that the intramolecular interaction between the pr imary binding loop and the scaffold of ECI plays an important role in the s trong inhibitory activity toward chymotrypsin. Furthermore, surface plasmon resonance analysis revealed that the side chains on the primary binding lo op of ECI contribute to both an increase in the association rate constant ( k(on)) and a decrease in the dissociation rate constant (k(off)) for the EC I-chymotrypsin interaction, whereas the backbone structure of the primary b inding loop mainly contributes to a decrease in the dissociation rate const ant.