STRUCTURE FUNCTION IMPLICATIONS FOR THE AMINOPEPTIDASE SPECIFICITY OFALEURAIN/

The cysteine protease aleurain, a member of the papain superfamily, wa s characterized by its specificity constants, k(cat)/K-m, for the hydr olysis of different substrates of the type H-P-1-NH-Mec (NH-Mec, 4-met hylcoumaryl-7-amide). The determined constants for the different subst rates decrease in the order citrulline > Arg = Phe >> Ala. A 75-fold l oss 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 pK(a1) and pK(a2) values of 5.81 and 7.27, respectively, at 25 degree s C. The residue corresponding to a pK(a1) value of 5.81 could be iden tified by its ionisation enthalpy, Delta H-ion, of 15 kJ/mol as a carb oxylate group of the enzyme interacting electrostatically with the res idue with pK(a2) 7.27, attributed to the alpha-amino group of the subs trate by its Delta N-ion value of 48 kJ/mol. Aleurain can be titrated at the active site with rans-epoxy-succinylleucylamido(4-guanidino)but ane, and the reaction was characterized by its association rate consta nt of 19000 M(-1).s(-1). Native chicken cystatin inhibited aleurain co mpetitively with K-i 133 nM. Recombinant chicken cystatin variants Ala -Glu-Phe-[Met(1), Ile(29), Leu(89)] chicken egg-white cystatin, (varia nt 1) and the N-terminally truncated form des-(S1-P11)-[Ala(12), Glu(1 2), Phe(14), Met(15), Ile(29), Leu(89)]-chicken egg-white cystatin, (v ariant 2), inhibited aleurain competitively with K-i values of 125 nM and 5 mu M, respectively. Implications for the aminopeptidase activity of aleurain are discussed using cathepsin H for comparison.