Kinetics of inhibition of bovine cathepsin S by bovine stefin B

The kinetics of the complex formation between bovine cathepsin S and bovine stefin B was studied by conventional and stopped-flow techniques. The inhibition at low inhibitor concentrations was tight and reversible (kass = 5.8 x 10(7) M-1.s-1, kdiss = 4.9 x 10(-4) s-1 at pH 6.0 and 25 degrees C), whereas at higher inhibitor concentrations it was pseudo-irreversible (kass = 6.14 x 10(7) M-1.s-1). The complex was formed directly lacking the fast pre-equilibrium step with the dissociation equilibrium constant of approximately 8 pM. The competitive nature of inhibition was confirmed. The kass was found to be pH-independent between pH 6.0 and 7.5 and decreased at lower or higher pH values in a way that strongly suggests involvement of two ionizable groups in the interaction (pKi = 5.2, pK2 = 8.3). The enzyme-substrate interaction seems to be influenced by different ionizable groups (pKi = 4.4, pK2 = 7.8).