JBC, Vol. 251, Issue 3, 754-762, Feb, 1976
Double-headed protease inhibitors from black-eyed peas. IV. Half-site reactivity in the formation of complexes with trypsin and chymotrypsin
L. S. Gennis and C. R. Cantor
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.
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