Second-order rate constants (cat/m) were first measured under pseudo-first-order conditions, that is, using a substrate concentration far below the m. cathepsin L mutant were further evaluated by using model components of the extracellular matrix, that is, Congo Red elastin, collagens I and II, and DQ-collagens I and IV. Results and Discussion Generation and expression of the S2 cathepsin K-like (Leu67Tyr/Ala205Leu) mutant of cathepsin L Molecular modeling studies and site-directed mutagenesis of cathepsin K have revealed some differences with cathepsin L in terms of size and shape of the side chains of their S2 pocket residues, especially in positions 67 and 205. Accordingly the S2 pocket of human cathepsin K was engineered into a cathepsin L-like variant (Y67L/L205A) (Lecaille et al. 2002b). A similar Rabbit Polyclonal to OR1A1 approach was used here to design the recombinant L67Y/A205L cathepsin L mutant. Briefly, the mutant was expressed in as a secreted zymogene (Mr 42,000) using the pPIC9K vector (see Fig. 1, lane 2), processed in the presence of pepsin at pH 4.0. After concentrating the supernatant (2 L) to a final volume of 15 mL, the L67Y/A205L cathepsin L variant was purified by chromatography (Butyl-Sepharose), which resulted in a single band of Mr 29,000 (Fig. 1, lane 3). Concentration of the purified mutant protein was 215 M (final volume = 200 L), as determined by E-64 titration. Open in a separate window Figure 1. SDS-PAGE (12% Tris-glycine) of the purified Leu67Tyr/Ala205Leu cathepsin L mutant. (Lane lane (Coomassie-blue staining). Characterization of L67Y/A205L cathepsin L mutant using fluorogenic substrates To examine Rhein-8-O-beta-D-glucopyranoside if the exchange of residues 67 and 205 of the S2 pockets of cathepsins K and L could induce a switch of their respective substrate specificity, the hydrolysis of two representative dipeptide substrates (Z-Phe-Arg-MCA, Z-Leu-Arg-MCA) and a tripeptide substrate (Z-Gly-Pro-Arg-MCA) was evaluated. Steady-state kinetics was performed as described in Materials and Methods (Br?mme et al. 1996), and the experimental data are summarized in Table 1. The values of cat and m were determined graphically from a Hanes linear plot, and their correctness confirmed by nonlinear regression analysis, while second-order rate constants (cat/m) were measured under pseudo-first-order conditions. In agreement with results of the profiling of the substrate specificity using positional scanning peptide libraries (Choe et al. 2006), wild-type cathepsin K exhibits a significant preference for Pro at P2 compared to wild-type cathepsin L, and mutations introduced in cathepsin K (Tyr67Leu/Leu205Ala) dramatically changed its S2 specificity (Lecaille et al. 2002b). Conversely, proline in Rhein-8-O-beta-D-glucopyranoside the P2 position is well accepted by the L67Y/A205L cathepsin L mutant with a m value identical to that of wild-type cathepsin K. Despite a fivefold lower second-order rate constant (due to a fivefold difference of cat value), when compared to that of cathepsin K, this result underlines the importance of Tyr67 in the S2 subsite for the accommodation of a proline residue at P2 (Aibe et al. 1996; Rhein-8-O-beta-D-glucopyranoside Xia et al. 1999; Smooker et al. 2000; Lecaille et al. 2002b). The mutation introduced in the S2 subsite of cathepsin L is also reflected by an inversion of the m values between Z-LR-MCA and Z-FR-MCA (Table 1). Unlike wild-type cathepsin L and the S2 mutant of cathepsin K (Y67L/L205A), which readily hydrolyze Z-Phe-Arg-MCA, the L67Y/A205L cathepsin L mutant prefers Z-Leu-Arg-MCA with an approximately sevenfold increase in cat/m. An analogous preference for this substrate is observed for wild-type cathepsin K. This difference in specificity for Leu in the P2 position was even greater for wild-type cathepsin L (22-fold reduction) and the Y67L/L205A cathepsin K mutant (35-fold reduction). Table 1. Hydrolysis of peptidyl-MCA substrates by Leu67Tyr/Ala205Leu cathepsin Rhein-8-O-beta-D-glucopyranoside L mutant, Tyr67Leu/Leu205Ala cathepsin K mutant, and wild-type cathepsins L and K Open in a separate window Inhibition by peptide-based inhibitors values depend, in fact, on differences between the buffers used for in vitro enzymatic assays (see Table 2). Its inhibitory profile Rhein-8-O-beta-D-glucopyranoside was reversed for the two mutants: Indeed, an 100-fold increase.