Inhibition of aminopeptidase activity by aromatic and other cyclic compounds

The effect of 2-naphthylamine, p-nitroaniline, o-phenanthroline, sodium deoxycholate and hydrocortisone succinate on the activity of human urine aminopeptidase, rat kidney methionyl and arginyl aminopeptidase, soybean and Enterolobium contortisiliquum seed aminopeptidase was studied using aminoacyl-2-naphthylamide and L-Leu-p-nitroanilide as substrates. Ki values ranged from 10 microM to 2.7 mM. On the basis of Ki and Km values, and catalytic efficiency for each enzyme, it is clear that the aminopeptidases from human urine and from soybean seed should be assayed with both substrates, whereas L-Leu-p-nitroaniline is a more appropriate substrate for the rat kidney aminopeptidases. Sodium deoxycholate is a better inhibitor than hydrocortisone succinate. Non-competitive inhibition was observed in all cases except for E. contortisiliquum seed aminopeptidase

Purification and partial characterization of Enterolobium contortisiliquum seed arylamidase

Arylamidase activity was isolated from Enterolobium contortisiliquum seeds (2 U/g) using L-Leu-2-naphthlamide as substrate to monitor the prification. The enzyme preparation was purified 733-fold by ammonium sulfate precipitation, and by ion eschange and gel filtration chromatography, in 6,6% yield. SDS-Polyacrylamide gel electrophoresis after fast protein liquid chromatography on a Mono Q column, showed only one protein band with a molecular weight of 35 kDa. The optimum pH for arylamidase activity was 6.5. Taking the hydrolysis rate of Lys-2-naphthylamide as one, the relative rates at which the other substrates were hydrolyzed were: Leu-2-naphthlamide, 30, Met-2-naphthlamide, 18, Arg-2-naphthlamide, 2, Ala-2-naphthylamide, 1.5, and L-Leu-p-nitroanilide, 26. The arylamidase activity was inhibited 50% by 0.1 mM HgCl2, 0.1 mM ZnCl2, 0.13 mM NiCl2, 0.2 mM o-phenanthroline and 1 * M soidum p-hydroxymercuribenzoate, and activated 35% by 5.0 * M EDTA. Iodoacetate (0.067 mM), dithioerythritol and 2-mercaptoethanol (3.3 mM), and chloride ions (0.2 M) had no effect on the enzyme activity