Probing cathepsin K activity with a selective substrate spanning its active site

The limited availability of highly selective cathepsin substrates seriously impairs studies designed to monitor individual cathepsin activities in biological samples. Among mammalian cysteine proteases, cathepsin K has a unique preference for a proline residue at P2, the primary determinant of its substrate specificity. Interestingly, congopain from Trypanosoma congolense also accommodates a proline residue in its S2 subsite. Analysis of a congopain model showed that amino acids forming its S2 subsite are identical with those of cathepsin K, except Leu⁶⁷ which is replaced by a tyrosine residue in cathepsin K. Furthermore, amino acid residues of the congopain S2′ binding pocket, which accepts a proline residue, are strictly identical with those of cathepsin K. Abz-HPGGPQ-EDN₂ph [where Abz represents o-aminobenzoic acid and EDN₂ph (= EDDnp) represents N-(2,4-dinitrophenyl)-ethylenediamine], a substrate initially developed for trypanosomal enzymes, was efficiently cleaved at the Gly-Gly bond by cathepsin K (k_cat/K_m = 426 000 M^-1 · s ^-1). On the other hand, Abz-HPGGPQ-EDN₂ph was resistant to hydrolysis by cathepsins B, F, H, L, S and V (20 nM enzyme concentration) and the Y67L (Tyr⁶⁷ → Leu)/L205 A cathepsin K mutant (20 nM), but still acted as a competitive inhibitor. Taken together, the selectivity of Abz-HPGGPQ-EDN₂ph to cathepsin K primarily depends on the S2 and S2′ subsite specificities of cathepsin K and the ionization state of histidine at P3. Whereas Abz-HPGGPQ-EDN₂ph was hydrolysed by wild-type mouse fibroblast lysates, its hydrolysis was completely abolished in the cathepsin K-deficient samples, indicating that Abz-HPGGPQ-EDN₂ph can be used to monitor selectively cathepsin K activity in physiological fluids and cell lysates.