The S2 subsites of cathepsins K and L and their contribution to collagen degradation

The exchange of residues 67 and 205 of the S2 pocket of human cysteine cathepsins K and L induces a permutation of their substrate specificity toward fluorogenic peptide substrates. While the cathepsin L‐like cathepsin K (Tyr67Leu/Leu205Ala) mutant has a marked preference for Phe, the Leu67Tyr/Ala205Leu cathepsin L variant shows an effective cathepsin K‐like preference for Leu and Pro. A similar turnaround of inhibition was observed by using specific inhibitors of cathepsin K [1‐(N‐Benzyloxycarbonyl‐leucyl)‐5‐(N‐Boc‐phenylalanyl‐leucyl)carbohydrazide] and cathepsin L [N‐(4‐biphenylacetyl)‐S‐methylcysteine‐(D)‐Arg‐Phe‐β‐phenethylamide]. Molecular modeling studies indicated that mutations alter the character of both S2 and S3 subsites, while docking calculations were consistent with kinetics data. The cathepsin K‐like cathepsin L was unable to mimic the collagen‐degrading activity of cathepsin K against collagens I and II, DQ‐collagens I and IV, and elastin‐Congo Red. In summary, double mutations of the S2 pocket of cathepsins K (Y67L/L205A) and L (L67Y/A205L) induce a switch of their enzymatic specificity toward small selective inhibitors and peptidyl substrates, confirming the key role of residues 67 and 205. However, mutations in the S2 subsite pocket of cathepsin L alone without engineering of binding sites to chondroitin sulfate are not sufficient to generate a cathepsin K‐like collagenase, emphasizing the pivotal role of the complex formation between glycosaminoglycans and cathepsin K for its unique collagenolytic activity.