Double-headed protease inhibitors from black-eyed peas. IV. Half-site reactivity in the formation of complexes with trypsin and chymotrypsin.
Complex formation between two new double-headed protease inhibitors from black-eyed peas, trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI), and trypsin and chymotrypsin was investigated in the concentration range from 10-8 to 10-4 M by titration experiments and gel filtration chromatography. Dissociation equilibrium constants measured for complexes detected in the titration experiments range from as large as 10-8 M for trypsin bound nonspecifically to the chymotrypsin site of BEPCI to as small as 10-18 M2 for the interaction of BEPCI with chymotrypsin. The identity and stoichiometry of complexes detected during titration experiments were confirmed by gel filtration of mixtures of native and fluorescently labeled proteases and inhibitors. Half-site reactivity is observed in the formation of complexes between BEPCI or BEPTI and trypsin and chymotrypsin at all experimentally practical concentrations. The double-headed complex contains 1 molecule each of trypsin, chymotrypsin, and BEPCI dimer. The bimolecular rate constants of complex formation between trypsin or chymotrypsin and isolated BEPCI oligomers range from 1.8 X 10(5) M-1 S-1 for chymotrypsin and BEPCI monomer to 4.4 X 10(7) M-1 S-1 for trypsin and the rapidly equilibrating BEPCI dimer. The estimated rate constants for the dissociation of half-site-liganded dimer complexes and liganded monomer complexes range from 7.5 X 10-3 S-1 for the trypsin-liganded BEPCI monomer complex to 1.6 X 10-6 S-1 for the chymotrypsin-liganded BEPCI dimer complex.
D001665:Binding Sites; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D005452:Fluoresceins; D007700:Kinetics; D046911:Macromolecular Substances; D010940:Plant Proteins / Q000494:pharmacology; D011480:Protease Inhibitors; D011485:Protein Binding; D012639:Seeds / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D014357:Trypsin; D014361:Trypsin Inhibitors
D005452:Fluoresceins; D046911:Macromolecular Substances; D010940:Plant Proteins; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; D002918:Chymotrypsin; D014357:Trypsin