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, k cat / K m , for the hydrolysis of different substrates of the type H‐P 1 ‐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 k cat / K m for H‐Arg‐NH‐Mec was bell‐shaped with p K a1 , and p K a2 values of 5.81 and 7.27, respectively, at 25°C. The residue corresponding to a p K a1 , value of 5.81 could be identified by its ionisation enthalpy, H ion , of 15 kJ/mol as a carboxylate group of the enzyme interacting electrostatically with the residue with p K a2 7.27, attributed to the α‐amino group of the substrate by its H ion , 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 19000 M −1 · s −1 . Native chicken cystatin inhibited aleurain competitively with K i 133 nM. Recombinant chicken cystatin variants Ala‐Glu‐Phe‐[Met 1 , Ile 29 , Leu 89 ] chicken egg‐white cystatin, (variant 1) and the N‐terminally truncated form des‐(S1‐P11)‐[Ala 12 , Glu 12 , Phe 14 , Met 15 , Ile 29 , Leu 89 ]‐chicken egg‐white cystatin, (variant 2), inhibited aleurain competitively with K i , values of 125 nM and 5 μM, respectively. Implications for the aminopeptidase activity of aleurain are discussed using cathepsin H for comparison.