| Abstrakt: |
The cysteine protease aleurain, a member of the papain superfamily, was characterized by its specificity constants, kcat/Km, for the hydrolysis of different substrates of the type H-P1-NH-Mec (NH-Mec, 4-methylcoumaryl-7-amide). The determined constants for the different substrates decrease in the order citrulline > Arg = Phc « Ala. A 75-fold loss of specificity was observed when the substrate Bz-Arg-NH-Mec (Bz, benzoyl), with a blocked N-terminus. was used instead of H-Arg-NH-Mec. The pH dependence of kcat/Km for H-Arg-NH-Mec was bell-shaped with pKa1 and pKa2 values of 5.81 and 7.27. respectively, at 25°C. The residue corresponding to a pKa1 value of 5.81 could be identified by its ionisation enthalpy, ΔHions, of 15 kJ/mol as a carboxylate group of the enzyme interacting electrostatically with the residue with pKa2 7.27. attributed to the α-amino group of the substrate by its ΔHion value of 48 kJ/mol. Aleurain can be titrated at the active sim with L-trans-epoxy-succinylleucylamido(4-guanidino)butane, and the reaction was characterized by its association rote constant of 19000 M-1 · s-1. Native chicken cystatin inhibited aleurain competitively with Ki 133 nM. Recombinant chicken cystatin variants Ala-Glu-Phe-[Met¹, Ile29, Leu89] Chicken egg-white cystatin, (variant 1) and the N-terminally truncated form des-(S1-P11)-[Ala12, Glu12, Phe14, Met15, Ile89]-chicken egg-white cystatin (variant 2), inhibited aleurain competitively with Ki values of 125 nM and 5 µM, respectively. Implications for the aminopeptidase activity of aleurain are discussed using cathepsin H for comparison. [ABSTRACT FROM AUTHOR] |
