Characterization of a Neutral Aminoacyl‐Peptide Hydrolase from Naegleria fowleri1

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Abstract
An intracellular alpha‐aminoacyl‐peptide hydrolase (EC 3.4.11.‐) from Naegleria fowteri nN68 (ATCC 30894) has been characterized. The enzyme preparation hydrolyzed phenylalanyl‐, tyrosyl‐, leucyl‐, arginyl‐, alanyl‐, tryptophanyl‐, histidyl‐, methionyl‐, and lysyl‐naphthylamide but not benzoylleucyl‐, leucylglycyl‐, glycylprolylleucyl‐, glycyl‐, threonyl‐, aspartyl‐, or glutamyl‐naphthylamide. The aminopeptidase activity was inhibited by the cysteine‐protease inhibitors—hydroxymercuribenzoate, chloromercurisulfate, and iodoacetate‐ by the aminopeptidase inhibitors‐bestatin and trans‐epoxysuccinyl‐leucyl‐agmatine‐ by an inhibitor of soluble alanyl aminopeptidase EC 3.4.11.14, puromycin, and by the metalloprotease inhibitor, o‐phenanthroline. The exopeptidase activity was not inhibited by the chelator, ethylenediaminetetraacetate, or the serine‐protease inhibitor, phenylmethylsulfonylfluoride. The pH optimum of the exopeptidase was between 7.0 and 8.0. Enzyme activity was stable at 55°C for 30 min, but all activity was lost after 15 min at 80°C. Enzyme activity was inhibited by 100 μM HgCI2 and CdCl2 but not by 1 mM CoCl2, CuCl2, MnCl2, NiCl2, FeCl2, or ZnCl2. Enzyme activity was inhibited by 0.1% sodium dodecyl sulfate but not by 0.2% Brij 35, Tween 20, Tween 80, or Triton X‐100.