title: Probing cathepsin K activity with a selective substrate spanning its active site.
abstract: 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/ K(m)=426000 M(-1) x 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.
authors.affiliation: 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.
authors.forename: Fabien
authors.initials: F
authors.lastname: Lecaille
authors.forename: Enrico
authors.initials: E
authors.lastname: Weidauer
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Dieter
authors.initials: D
authors.lastname: Brömme
authors.forename: Gilles
authors.initials: G
authors.lastname: Lalmanach
mesh_terms: D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D056657:Cathepsin K; D002403:Cathepsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012867:Skin / Q000166:cytology / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014348:Trypanosoma congolense / Q000201:enzymology
chemical_list: D009842:Oligopeptides; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C084086:congopain; C534130:CTSK protein, human; D056657:Cathepsin K; C534132:Ctsk protein, mouse