POTENT SLOW-BINDING INHIBITION OF CATHEPSIN-B BY ITS PROPEPTIDE

A peptide (PCB1) corresponding to the proregion of the rat cysteine protease cathepsin B was synthesized and its ability to inhibit cathepsin B activity investigated. PCB1 was found to be a potent inhibitor of mature cathepsin B at pH 6.0, yielding a K(i) = 0.4 nM. This inhibition obeyed slow-binding kinetics and occurred as a one-step process with a k1 = 5.2 x 10(5) M-1 s-1 and a k2 = 2.2 x 10(-4) s-1. On dropping from pH 6.0 to 4.7, K(i) increased markedly, and whereas k1 remained essentially unchanged, k2 increased to 4.5 x 10(-3) s-1. Thus, the increase in K(i) at lower pH is due primarily to an increased dissociation rate for the cathepsin B/PCB1 complex. At pH 4.0, the inhibition was 160-fold weaker (K(i) = 64 nM) than at pH 6.0, and the propeptide appeared to behave as a classical competitive inhibitor rather than a slow-binding inhibitor. Incubation of cathepsin B with a 10-fold excess of PCB1 overnight at pH 4.0 resulted in extensive cleavage of the propeptide whereas no cleavage occurred at pH 6.0, consistent with the formation of a tight complex between cathepsin B and PCB1 at the higher pH. The synthetic propeptide of cathepsin B was found to be a much weaker inhibitor of papain, a structurally similar cysteine protease, and no pH dependence was observed. Inhibition constants of 2.8 and 5.6 muM were obtained for papain inhibition by PCB1 at pH 4.0 and 6.0, respectively. Incubation with a 10-fold excess of the propeptide resulted in its cleavage by papain at both pH values. The results presented here complement the data from similar investigations with serine and aspartic proteases and with metalloproteases indicating that the proregion of all four classes of protease plays an important physiological role in inactivating their corresponding enzyme.