The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

Leupeptin was found to be a slow, tight-binding inhibitor of cathepsin B from human spleen and rabbit liver. During the enzyme-catalyzed reaction in the presence of inhibitor a concentration-dependent transient state, lasting several minutes, preceded the attainment of the steady state and was characterized by a concave upward or a concave downward lag phase depending on whether the enzyme had been preincubated with the inhibitor or not, respectively. From the pre-steady-state phase of the curves both k on and k off for the formation of the enzyme-inhibitor complex could be calculated. Ki, as the ratio k off/k on, was in good agreement with the inhibition constant obtained using a steady-state treatment. k on was 1.8 X 10(5) M-1 s-1 and 2.0 X 10(5) M-1 s-1 for the human and rabbit enzyme, respectively and the slowness of the binding process fitted into the general concept of enzyme hysteresis. The activation of the essential cysteine residue of cathepsin B by dithiothreitol was also a very slow process characterized by a second-order rate constant of 4.1 M-1 s-1. The kinetic features of leupeptin binding allow the prediction of the possible efficiency of this inhibitor on cathepsin B in vivo. It is shown that in order for leupeptin to be a physiologically significant inhibitor of cathepsin B, its concentration at the target site must exceed 10 microM, at least. This contrasts with the predictions drawn from the value of Ki (approximately 5 nM), which would suggest an effective inhibition of the enzyme already at a concentration of 0.05 microM.