FIGURE 2.

Design of a CatS-specific substrate. HPLC analysis of three representative substrates at a concentration of 20 μm after incubation with 1.3 nm CatS (left column), 11.6 nm CatB (middle column), and 13 nm CatL (right column) for 1 h in digestion buffer at pH 5.5 and 37 °C. Digestion products were detected by their UV absorbance at 220 nm. The black lines represent the chromatograms of the digestion mixtures, whereas gray lines are chromatograms of the undigested negative controls. A, digestion analysis of the unspecific substrate KVSVR. The substrate is completely digested by CatB and CatL and only partially by CatS within the given time. The fragments Mca-GRWHKVS (peak 1) and VRWE-Lys(Dnp)-DArg-NH₂ (peak 3) of Mca-GRWHKVSVRWE-Lys(Dnp)-DArg-NH₂ (peak 4) are generated by all three enzymes. CatS additionally generates cleavage products Mca-GRWHKVSVR (peak 2) and WE-Lys(Dnp)-DArg-NH₂ (peak 3). B, digestion analysis of the basis peptide TVGLR, which was used in the library experiment. Compared with KVSVR the substrate is now completely digested by CatS and only partially by CatB and CatL. Although the substrate Mca-GRWHTVGLRWE-Lys(Dnp)-DArg-NH₂ (peak 3) shows only one major CatS cleavage site and the corresponding fragments Mca-GRWHTVG (peak 1) and LRWE-Lys(Dnp)-DArg-NH₂ (peak 2) are present in the reaction mixture of all three enzymes, CatB and CatL generate additional cleavage products. C, digestion analysis of the CatS specific substrate PMGLP. This substrate is strongly digested by CatS and on the other hand completely resistant against CatB and CatL digestion within the given time. CatS cleaves the substrate Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH₂ (peak 3) between G and L. The generated fragments Mca-GRWPPMG (peak 1) and LPWE-Lys(Dnp)-DArg-NH₂ (peak 2) elute at the same retention time. During incubation the methionine residue is oxidized leading to oxidation peaks (Ox).