Dissociation of ionizing groups in the binding cleft inversely controls the endo- and exopeptidase activities of cathepsin B.

Abstract

Negatively charged reactants are sensitive reactivity probes of the active site of cysteine proteases (Halász, P., and Polgár, L. (1977) Eur. J. Biochem. 79, 491-494). Thus, the thiolate-imidazolium ion pair of papain reacts at an enhanced rate with iodoacetate due to a favorable interaction between the positive imidazolium ion of the ion pair and the negative carboxylate of the alkylating agent. We have found that cathepsin B, the closely related lysosomal cysteine protease, also shows enhanced reactivity toward iodoacetate, indicating the presence of the catalytically competent thiolate-imidazolium ion pair in this enzyme. However, the pH dependence of the reaction is different. Papain exhibits a simple bell-shaped curve, whereas cathepsin B exhibits a complex pH dependence which is controlled by an ionizing group with a pKa of about 5.5. This finding indicates the existence of two reactive forms associated with the active site of cathepsin B: a high reactivity form below pH 5.5 and a low reactivity form above pH 5.5. The former accounts for the exopeptidase (peptidyl dipeptidase) activity of the enzyme and the latter for the endopeptidase activity, measured with the highly specific substrate benzyloxycarbonyl-Arg-Arg-2-naphthylamide. As seen from active site models, cathepsin B, in contrast to papain, contains a triad of charged groups near the thiolate-imidazolium ion pair which is composed of Glu-131, Arg-162, and Glu-205. A net negative charge above pH 5.5 and the positive charge of Arg-162 below pH 5.5 may control the exo- and endopeptidase activities, as well as the alkylation with iodoacetate. This can be mediated through electrostatic interactions with the charged reactants and, possibly, also by causing a conformational change in the geometry of the thiolate-imidazolium ion pair.