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

¹ INSERM, U 618, Tours, F-37000, France
² Université François Rabelais, Tours, F-37000, France
³ IFR 135, Tours, F-37000, France
⁴ Lady Davis Institute of Medical Research, Jewish General Hospital, McGill University Health Centre, Montreal, Canada
⁵ Computational Chemistry, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada
⁶ Department of Dentistry and UBC Centre for Blood Research, University of British Columbia, Vancouver, Canada

(RECEIVED November 15, 2006; FINAL REVISION January 12, 2007; ACCEPTED January 12, 2007)

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--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.

Keywords: cathepsin; collagen; cysteine protease; peptide inhibitor; substrate specificity

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