An exploration of the primary specificity site of cathepsin B.

Peptidyl diazomethyl ketones inactivate cathepsin B apparently by alkylation of the active center thiol following complex formation as in the case of benzyloxycarbonyl (Cbz)-Phe-AlaCHN2. The phenylalanine contributes considerably to binding in the secondary specificity site. In order to define the topography of the active center region comprising the primary specificity site of beef spleen cathepsin B, a series of peptidyl diazomethyl ketones having the general structure Cbz-Phe-X-CHN2 has now been synthesized. The amino acid, X, has been varied in size to include rather large side chains which might reveal available binding potential or limitations. Some of the reagents, in fact, were not inhibitory even at 10(-4) M. Others, however, that did measurably inactivate cathepsin B provided a range of reactivities that extended over 5 orders of magnitude and correlated with affinity in the reversible phase of inactivation. Some large side chains, for example, that of tryptophan, were very poorly tolerated in this region of the active center, whereas others, such as O-benzyl threonine, provided remarkably active inhibitors. A topographical rationalization of the results is offered.