Substrate specificity and binding loci for inhibitors in an aminopeptidase purified from the plasma membrane of midgut cells of an insect (Rhynchosciara americana) larva.
Plasma membrane-bound aminopeptidases (EC 3.4.11.2) are found in the midgut cells from Rhynchosciara americana larvae, and are recovered in soluble form after papain treatment. The major papain-released aminopeptidase (Mr 207,000 and pI 7.8) was shown to be a true aminopeptidase with a broad specificity toward aminoacyl-beta-naphthylamides and to be more active on tetra and tripeptides than on dipeptides. The purified aminopeptidase is inactivated by EDTA according to a kinetics which is half order in relation to EDTA. Leucine hydroxamate (Ki 27 microM) and hydroxylamine (Ki 5.4 mM) completely protect the enzyme from inactivation by EDTA, whereas isoamyl alcohol (Ki 62 mM) increases the inactivation rate. There are 2.3 binding sites in the enzyme for phenanthroline, which makes the binding of the substrate in the enzyme difficult, changes the enzyme-substrate into a more productive complex, and increases the inactivation rate of the enzyme by EDTA by 87-fold. The data support the proposal that the enzyme has a metal ion which is catalytically active and that the enzyme displays two subsites in its active center: a hydrophobic subsite, to which isoamyl alcohol binds exposing the metal ion, and a polar subsite, to which hydroxylamine binds.
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D000626:Aminopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004175:Diptera / Q000201:enzymology*; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology; D007700:Kinetics; D007814:Larva; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D010206:Papain / Q000032:analysis; D010618:Phenanthrolines / Q000494:pharmacology; D013379:Substrate Specificity
D010618:Phenanthrolines; C032796:leucine hydroxamate; D004492:Edetic Acid; D000626:Aminopeptidases; D010206:Papain; D007930:Leucine