Kinetics of inhibition of bovine cathepsin S by bovine stefin B

Abstract

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 (k _ass = 5.8 × 10⁷ M⁻¹ · s⁻¹, k _diss = 4.9 × 10⁻⁴ s⁻¹ at pH 6.0 and 25°C), whereas at higher inhibitor concentrations it was pseudo-irreversible (k _ass = 6.14 × 10⁷ M⁻¹ · s⁻¹). The complex was formed directly lacking the fast preequilibrium step with the dissociation equilibrium constant of ~ 8 pM. The competitive nature of inhibition was confirmed. The k _ass 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 (pK ₁ = 5.2, pK ₂ = 8.3). The enzyme-substrate interaction seems to be influenced by different ionizable groups (pK ₁ = 4.4, pK ₂ = 7.8).