KINETICS OF INHIBITION OF BOVINE CATHEPSIN-S BY BOVINE STEFIN-B

The kinetics of the complex formation between bovine cathepsin S and b ovine stefin B was studied by conventional and stopped-flow techniques . The inhibition at low inhibitor concentrations was tight and reversi ble (k(ass) = 5.8 x 10(7) M(-1) . s(-1), k(diss) = 4.9 x 10(-4) s(-1) at pH 6.0 and 25 degrees C), whereas at higher inhibitor concentration s it was pseudo-irreversible (k(ass) = 6.14x 10(7) M(-1) . s(-1)). The complex was formed directly lacking the fast pre equilibrium step wit h the dissociation equilibrium constant of similar to 8 pM. The compet itive nature of inhibition was confirmed. The k(ass) was found to be p H-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-subs trate interaction seems to be influenced by different ionizable groups (pK(1) = 4.4, PK2 = 7.8).