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 (kcat/Km=426000 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)/L205A 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.
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October 2003
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Research Article|
October 15 2003
Probing cathepsin K activity with a selective substrate spanning its active site
Fabien LECAILLE;
Fabien LECAILLE
∗INSERM EMI-U 00-10 ‘Protéases et Vectorisation’, Laboratoire d'Enzymologie et Chimie des Protéines, Faculté de Médecine, Université François Rabelais, 2 bis, Boulevard Tonnellé, F-37032 Tours Cedex, France
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Enrico WEIDAUER;
Enrico WEIDAUER
†Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, U.S.A.
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Maria A. JULIANO;
Maria A. JULIANO
‡Escola Paulista de Medicina, Universidade Federal de São Paulo, 04044-020 São Paulo, Brazil
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Dieter BRÖMME;
Dieter BRÖMME
†Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, U.S.A.
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Gilles LALMANACH
Gilles LALMANACH
1
∗INSERM EMI-U 00-10 ‘Protéases et Vectorisation’, Laboratoire d'Enzymologie et Chimie des Protéines, Faculté de Médecine, Université François Rabelais, 2 bis, Boulevard Tonnellé, F-37032 Tours Cedex, France
1To whom correspondence should be addressed (e-mail lalmanach@univ-tours.fr).
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Publisher: Portland Press Ltd
Received:
March 26 2003
Revision Received:
June 27 2003
Accepted:
July 01 2003
Accepted Manuscript online:
July 01 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 375 (2): 307–312.
Article history
Received:
March 26 2003
Revision Received:
June 27 2003
Accepted:
July 01 2003
Accepted Manuscript online:
July 01 2003
Citation
Fabien LECAILLE, Enrico WEIDAUER, Maria A. JULIANO, Dieter BRÖMME, Gilles LALMANACH; Probing cathepsin K activity with a selective substrate spanning its active site. Biochem J 15 October 2003; 375 (2): 307–312. doi: https://doi.org/10.1042/bj20030468
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