1979 Volume 85 Issue 2 Pages 601-607
The present paper deals with the inhibition and the binding of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), against α-chymotrypsin. Both α-chymotrypsin and trypsin have been reported not to be inhibited by this inhibitor when casein is used as a substrate (Sato, S. and Murao, S. (1973) Agric. Biol. Chem. 37, 1067-1074). However, in this study, SSI was shown to inhibit α-chymotrypsin competitively with an inhibitor constant, K1=3.8μM, by using p-nitrophenyl acetate as a substrate. The dissociation constant of α-chymotrypsin-SSI complex determined by static titration utilizing the small ultraviolet absorption difference spectra observed on the binding of the enzyme and SSI (Kd) is 2.2μM, and that obtained utilizing the visible absorption difference spectra of proflavine bound to the active site of the enzyme (Kd') is 2.9μM. These values are almost 104 times larger than the inhibitor constant estimated for subtilisin BPN', which was of the order of 10-10M (Inouye, K. et al. (1977) J. Biochem. 82, 961-967).
It was demonstrated by spectrophotometric titration and equilibrium dialysis that proflavine bound at the active site of α-chymotrypsin is not displaced by SSI binding, but forms a ternary complex with the enzyme and the inhibitor. The dissociation constant of the α-chymotrypsin-proflavine complex into the enzyme and proflavine (Kp) was determined to be 51±1μM by spectrophotometric titration and 64±5μM by equilibrium dialysis; the dissociation constant of proflavine from α-chymotrypsin-SSI-proflavine complex (Kp') was determined to be 132±8μM by spectrophotometric titration and 160±8μM by equilibrium dialysis.
