Analysis of the Complex Formed by Erythrina variegata Chymotrypsin Inhibitor with Chymotrypsin and Properties of the Peptides Prepared from the Inhibitor by a Limited Proteolysis

Abstract

The stoichiometry of Erythrina vuriegata chymotrypsin inhibitor (ECI) and chymotrypsin interaction was previously estimated to be 1 : 2 by a titration of inhibitory activity. In the present study, gel-permeation chromatography and reverse-phase HPLC (RP-HPLC) were employed to analyze the complex formed by the inhibitor and enzyme. The results showed that ECI and chymotrypsin molecules undergo aggregation in the complex-forming buffer simultaneously with a binary complex consisting of one ECI and one chymotrypsin molecules in a soluble form.A mild lysylendopeptidase digestion of ECI produced two peptides in high yield, which were separated by RP-HPLC and characterized in terms of their structures and inhibitory activities. The N-terminal peptide, ECI-( 1 -107)-peptide, containing the primary reactive site retained a slight inhibitory activity, while the C-terminal peptide, ECI-( 108 -179)-peptide, exhibited no inhibitory activity. The inhibitory potency of the ECI-(l-107)-peptide was enhanced by the presence of the ECI-(108-179)-peptide in reconstituted mixture. Recovery of the native-like structure of the reconstituted complex was further indicated by fluorescence spectra, which showed strong conformational interaction between the two peptides ; their dissociation constant Kd was calculated to be 209 nM. Taken together with the previous result obtained by chymotryptic digestion, it is suggested that the primary binding loop in ECI interacts with chymotrypsin not only by a standard mechanism but also by a non-substrate-like manner. Alternatively, ECI might have an additional binding segment in the N-terminal region which interacts with chymotrypsin by a non-substrate-like manner. Further, it is shown that the C-terminal region may support the native conformation of the binding loop(s) in the N-terminal region as an intramolecular chaperone.Keywords: chymotrypsin inhibitor; Erythrina variegutu ; Kunitz-family protein.Since Sweet et al. elucidated the tertiary structure of a complex consisting of soybean trypsin inhibitor (STI) and porcine trypsin [I], a vast amount of structural information has been accumulated for protein structures of plant serine proteinase inhibitors [2]. The crystal structures of these complexes with the cognate proteinases indicate that they all possess an exposed binding loop of a characteristic canonical conformation which associates with the catalytic residues in a similar manner to that of productively bound substrates. Thus, these studies have provided valuable insights into the nature of protein-protein interaction and also into the catalytic mechanism of serine proteinase.Apart from structural studies on plant proteinase inhibitors, the current interest in plant proteinase inhibitors is their application for transgenic plants with resistances to insects and pathogenes. Several positive results employing potato inhibitors I and I1 [3] and cowpea inhibitors [4] were reported. However, a recent study showed that Spodoptera exigua larvae adapt to the exogenous proteinase inhibitor by indu...