In:
European Journal of Biochemistry, Wiley, Vol. 249, No. 3 ( 1997-11), p. 870-877
Abstract:
The stoichiometry of Erythrina variegata 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‐(1–107)‐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 K d 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.
Type of Medium:
Online Resource
ISSN:
0014-2956
,
1432-1033
DOI:
10.1111/ejb.1997.249.issue-3
DOI:
10.1111/j.1432-1033.1997.t01-1-00870.x
Language:
English
Publisher:
Wiley
Publication Date:
1997
detail.hit.zdb_id:
1398347-7
detail.hit.zdb_id:
2172518-4
SSG:
12
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