title: Biochemical characterization of human cathepsin X revealed that the enzyme is an exopeptidase, acting as carboxymonopeptidase or carboxydipeptidase.
abstract: Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of approximately 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 approximately 5.0 x 103 M-1.s-1) were degraded approximately 25-fold less efficiently than the carboxypeptidase substrates (kcat/Km approximately 120.0 x 103 M-1.s-1).
authors.affiliation: Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
authors.forename: I
authors.initials: I
authors.lastname: Klemencic
authors.forename: A K
authors.initials: AK
authors.lastname: Carmona
authors.forename: M H
authors.initials: MH
authors.lastname: Cezari
authors.forename: M A
authors.initials: MA
authors.lastname: Juliano
authors.forename: L
authors.initials: L
authors.lastname: Juliano
authors.forename: G
authors.initials: G
authors.lastname: Guncar
authors.forename: D
authors.initials: D
authors.lastname: Turk
authors.forename: I
authors.initials: I
authors.lastname: Krizaj
authors.forename: V
authors.initials: V
authors.lastname: Turk
authors.forename: B
authors.initials: B
authors.lastname: Turk
mesh_terms: D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000302:isolation & purification / Q000378:metabolism*; D056657:Cathepsin K; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D013379:Substrate Specificity
chemical_list: D002268:Carboxypeptidases; D002403:Cathepsins; D010450:Endopeptidases; C534130:CTSK protein, human; D056657:Cathepsin K