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 chymotrypsin 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 that the stoichiometry of ECI and chymotrypsin was 1:1, though the apparent molecular mass of the complex was estimated to be 60 kDa. To examine the contribution of Leu64 (the P1 residue) to the inhibitory activity of ECI, a complete set of mutated inhibitors in which the amino acid at position 64 was replaced by 19 other amino acid residues was constructed by means of site-directed mutagenesis. Potent inhibitory activities (Ki, 1.3-4.6 x 10(-8) M) exceeding that of the wild-type ECI (Ki, 9.8 x 10(-8) M) were present in the mutant proteins L64F, L64M, L64W, and L64Y. The inhibitory activity of the mutant L64R was practically identical to that of the wild-type ECI. All other mutants exhibited slightly decreased inhibitory activities with Ki values of 1.9-4.6 x 10(-7) M. These results indicate that ECI-chymotrypsin interaction involves not only the P1 site residue but also other residue(s) of ECI. A series of individual alanine mutations was then constructed in residues Gln62 (P3), Phe63 (P2), Ser65 (P1'), Thr66 (P2'), and Phe67 (P3') in order to evaluate the contribution of each residue in the primary binding loop to the inhibitory activity. Replacement of Gln62, Phe63, and Phe67 with Ala residues decreased the inhibitory activity, the Ki values being increased by approximately 3-4-fold; but replacement of Ser65 and Thr66 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 chymotrypsin.
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581, Japan.
S
S
Iwanaga
N
N
Yamasaki
M
M
Kimura
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D010944:Plants / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D017931:DNA Primers; D014361:Trypsin Inhibitors; D002918:Chymotrypsin