Interaction of α-Chymotrypsin and a Protein Proteinase Inhibitor, Streptomyces Subtilisin Inhibitor

Abstract

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=3·8 μM p 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 (K_d) is 2·2 μM, and that obtained utilizing the visible absorption difference spectra of proflavine bound to the active site of the enzyme (K_d') is 2·9 μM These values are almost 10⁴ times larger than the inhibitor constant estimated for subtilisin BPN', which was of the order of 10⁻¹⁰ M (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 a-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 (K_p) was determined to be 51 ±1 μM by spectrophotometric titration and 64±5 μM by equilibrium dialysis; the dissociation constant of proflavine from α-chymotrypsin-SSI-proflavine complex (K_p) was determined to be 132 ±8 μM by spectrophotometric titration and 160±8 μM by equilibrium dialysis.