Structure/function implications for the aminopeptidase specificity of aleurain.
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 = Phe >> 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 degrees C. The residue corresponding to a pKa1 value of 5.81 could be identified by its ionisation enthalpy, delta Hion, of 15 kJ/mol as a carboxylate group of the enzyme interacting electrostatically with the residue with pKa2 7.27, attributed to the alpha-amino group of the substrate by its delta Hion value of 48 kJ/mol. Aleurain can be titrated at the active site with L-trans-epoxy-succinylleucylamido(4-guanidino)butane, and the reaction was characterized by its association rate constant of 19,000 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, Leu89]-chicken egg-white cystatin, (variant 2), inhibited aleurain competitively with Ki values of 125 nM and 5 microM, respectively. Implications for the aminopeptidase activity of aleurain are discussed using cathepsin H for comparison.
Rothe
M
M
Institut für Biochemie, Technischen Hochschule Darmstadt, Germany.
Zichner
A
A
Auerswald
E A
EA
Dodt
J
J
D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D015891:Cystatins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology; D007700:Kinetics; D010206:Papain / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D015891:Cystatins; D011994:Recombinant Proteins; D000626:Aminopeptidases; D003546:Cysteine Endopeptidases; C046726:aleurain; D010206:Papain