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,, and k o f f for the formation of the enzyme-inhibitor complex could be calculated. Ki, as the ratio koff/kon, was in good agreement with the inhibition constant obtained using a steady-state treatment. k,, was 1.8 x lo5 M-' s p l and 2 . 0~ lo5 M-' s-' 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 -' S -' . 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 20 pM, at least. This contrasts with the predictions drawn from the value of K , ( z 5 nM), which would suggest an effective inhibition of the enzyme already at a concentration of 0.05 pM.Leupeptins are peptide derivatives isolated from culture filtrates of some Strqitomjxvs species [I, 21 and identified as mixtures of N-u-acyl-tripeptides containing the unusual amino aldehyde arginal at the C-terminal position. Leupeptins are potent competitive inhibitors of cysteine proteinases such as cathepsin B and papain [1,3] and serine proteinases such as trypsin, plasmin, kallikrein and acrosin [4,5].Cathepsin B is a lysosomal proteinase containing an essential cysteine residue in the active center and is found in the tissues of many animal species 161. While the normal physiological function of cathepsin B may be the intracellular breakdown of proteins [7,8], an implication of this enzyme has been proposed also in some pathological situations as cartilage destruction 191, tumor invasion [lo, 1 I] and muscular dystrophy [I 21.Recent work in this laboratory on the role of extracellular proteinases in the processes of tissue destruction in rheumatoid arthritis 1131 and tumor invasion [11,14] led us to the study of the inhibition mechanisms of these enzymes by some agents of established therapeutic significance [I5 -171. We have undertaken the present investigation on the inhibition of cathepsin B by leupeptin in virro in order to gain more information on the properties of the system before attempting experiments in vivo.The present paper reports on: (a) the purification of human spleen cathepsin B ; (b) the theory of tight-binding inhibition as a necessary complement to that which is k...