Biochemical characterization of human cathepsin X revealed that the enzyme is an exopeptidase, acting as carboxymonopeptidase or carboxydipeptidase

Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of ≈ 33 kDa and pI 5.1–5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (Ki = 1.7–15.0 nm), but poorly or not at all by stefin B (Ki > 250 nm) and l‐kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA‐074 and GFG‐semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1′ position, although the enzyme cleaved all P1′ residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin X also cleaved substrates with amide‐blocked C‐terminal carboxyl group with rates similar to those of the unblocked substrates. In contrast, no endopeptidase activity of cathepsin X could be detected on a series of o‐aminobenzoic acid‐peptidyl‐N‐[2,‐dinitrophenyl]ethylenediamine substrates. Furthermore, the standard cysteine protease methylcoumarine amide substrates (kcat/Km≈ 5.0 × 103 m−1·s−1) were degraded ≈ 25‐fold less efficiently than the carboxypeptidase substrates (kcat/Km ≈ 120.0 × 103 m−1·s−1).