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.

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概要

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• We previously demonstrated that amino acid residues Gln62 (P3), Phe63 (P2), Leu64 (P1), and Phe67 (P3') in the primary binding loop of Erythrina variegata chymotrypsin inhibitor (ECI), a member of the Kunitz inhibitor family, are involved in its strong inhibitory activity toward chymotrypsin [Iwanaga et al. (1998) J. Biochem. 124, 663-669]. To determine whether or not these four amino acid residues predominantly contribute to the strong inhibitory activity of ECI, they were simultaneously replaced by Ala. The results showed that a quadruple mutant, Q62A/F63A/L64A/F67A, retained considerable inhibitory activity (K1, 5.6×10-7M), indicating that in addition to the side chains of these four amino acid residues, the backbone structure of the primary binding loop in ECI is essential for the inhibitory activity toward chymotrypsin. Two chimeric proteins, in which the primary binding loops of ECI and ETIa were exchanged: an isoinhibitor from E. variegata with lower 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 chimeric protein, ECI/ETIa, composed of the primary binding loop of ECI and the scaffold of ETIa showed weaker inhibitory activity (K1, 1.3×10-6M) than ECI (K1, 9.8×10-8 M). In contrast, a chimera, ETIa/ECI, comprising the primary binding loop of ETIa and the scaffold of ECI inhibited chymotrypsin more strongly (K1, 5.7×10-7M) than ETIa (K1, 1.3×10-6M). These results indicate that the intramolecular interaction between the primary binding loop and the scaffold of ECI plays an important role in the strong inhibitory activity toward chymotrypsin. Furthermore, surface plasmon resonance analysis revealed that the side chains on the primary binding loop of ECI contribute to both an increase in the association rate constant (kon) and a decrease in the dissociation rate constant (koff) for the ECI-chymotrypsin interaction, whereas the backbone structure of the primary binding loop mainly contributes to a decrease in the dissociation rate constant.

• 社団法人 日本生化学会の論文

著者

• KOUZUMA Yoshiaki

  Laboratory of Biochemistry, Faculty of Agriculture, Graduate School, Kyushu University

• Miyamoto Atsushi

  Laboratories Of Veterinary Pharmacology

• Iwanaga Shiroh

  Laboratory Of Biochemistry Faculty Of Agriculture Graduate School Kyushu University

• Kouzuma Yoshiaki

  Laboratory Of Food Functionality School Of Agriculture Ibaraki University

• Nagata Ruby

  Laboratory Of Biochemistry Faculty Of Agriculture Kyushu University

• Yamasaki Nobuyuki

  Laboratory Of Agricultural Process Engineering Faculty Of Agriculture Kyushu University

• KOUZUMA Yoshiaki

  Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University

• KIMURA Makoto

  Laboratory for Remediation Research, Plant Science Center, RIKEN

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