title: Studies on the aminopeptidase activity of rat cathepsin H.
abstract: Three synthetic substrates H-Arg-NH-Mec, Bz-Arg-NH-Mec and H-Cit-NH-Mec (Bz, Benzoyl; NH-Mec, 4-methylcoumaryl-7-amide; Cit, citrulline) were used to characterize specificity requirements for the P1-S1 interaction of cathepsin H from rat liver. From rapid equilibrium kinetic studies it was shown that Km, kcat and the specificity constants kcat/Km are quite similar for substrates with a free alpha-amino group. In contrast, a 25-fold decrease of kcat/Km was observed for the N-terminal-blocked substrate Bz-Arg-NH-Mec. The activation energies for H-Arg-NH-Mec and Bz-Arg-NH-Mec were determined to be 37 kJ/mol and 55 kJ/mol, respectively, and the incremental binding energy delta delta Gb of the charged alpha-amino group was estimated to -8.1 kJ/mol at pH 6.8. The shown preference of cathepsin H for the unblocked substrates H-Arg-NH-Mec and H-Cit-NH-Mec was further investigated by inspection of the pH dependence of kcat/Km. The curves of the two substrates with a charged alpha-amino group showed identical bell-shaped profiles which both exhibit pKa1 and pKa2 values of 5.5 and 7.4, respectively, at 30 degrees C. The residue with a pKa1 of 5.5 in the acid limb of the activity profile of H-Arg-NH-Mec was identified by its ionization enthalpy delta Hion = 21 kJ/mol as a beta-carboxylate or gamma-carboxylate of the enzyme, whereas the residue with a pKa2 of 7.4 was assigned to the free alpha-amino group of the substrate with a delta Hion of 59 kJ/mol. Bz-Arg-NH-Mec showed a different pH-activity profile with a pKa1 of 5.4 and a pKa2 of 6.6 at 30 degrees C. Cathepsin H exhibits no preference for a basic P1 side chain as has been shown by the similar kinetics of H-Arg-NH-Mec and the uncharged, isosteric substrate H-Cit-NH-Mec. In summary, specific interactions of an anionic cathepsin H active site residue with the charged alpha-amino group of substrates caused transition state stabilization which proves the enzyme to act preferentially as an aminopeptidase.
authors: Institut für Biochemie der Technischen Hochschule Darmstadt, Federal Republic of Germany.
authors: M
authors: M
authors: Rothe
authors: J
authors: J
authors: Dodt
mesh_terms: D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D002151:Calorimetry; D056655:Cathepsin H; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D051381:Rats; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D002403:Cathepsins; D000626:Aminopeptidases; D003546:Cysteine Endopeptidases; D056655:Cathepsin H; C534128:Ctsh protein, rat