title: Synergistic inhibition of the enzymatic activity of aminopeptidase N by divalent metal ion chelators.
abstract: Membranes of HEK293 cells that were transfected with human aminopeptidase N (AP-N, CD13, EC 3.4.11.2) and purified soluble porcine kidney AP-N were used to study inhibition of its enzyme activity by divalent cation chelators. Whereas pre-incubation for 10 min with ethylenediaminetetraacetic acid (EDTA), did not or only weakly affected the enzyme activity, the bidentate chelator 1,10-phenanthroline produced a complete and concentration-dependent inhibition of AP-N. The corresponding curves had Hill slopes of 2.50 +/- 0.23 and 2.73 +/- 0.01 for soluble and recombinant AP-N respectively. EDTA increased the potency of 1,10-phenanthroline till a limit, at which Hill slopes became close to unity. In the absence of EDTA, the inhibition by 1,10-phenanthroline was only weakly affected by the substrate concentration. On the other hand, competition between 1,10-phenanthroline and the substrate took place in the presence of EDTA. Similar findings were reported for the related metallopeptidase cystinyl aminopeptidase and point towards a model in which 1,10-phenanthroline inhibit enzyme activity by decreasing the free Zn2+ concentration. Moreover, EDTA is capable of removing a modulatory ion from an allosteric site at the enzyme, facilitating the direct interaction between 1,10-phenanthroline and the catalytic Zn2+. Compatible with this model, Ca2+ may bind to this allosteric site resulting in the potentiation of Zn2+-mediated re-activation of the enzyme activity in the presence of EDTA and 1,10-phenanthroline.
authors.affiliation: Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. pvandhey@vub.ac.be
authors.forename: Patrick M L
authors.initials: PM
authors.lastname: Vanderheyden
authors.forename: Heidi
authors.initials: H
authors.lastname: Demaegdt
authors.forename: Julie
authors.initials: J
authors.lastname: Swales
authors.forename: Pieter-Jan
authors.initials: PJ
authors.lastname: Lenaerts
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: De Backer
authors.forename: Lotte K
authors.initials: LK
authors.lastname: Vogel
authors.forename: Georges
authors.initials: G
authors.lastname: Vauquelin
mesh_terms: D000818:Animals; D018826:CD13 Antigens / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D002614:Chelating Agents / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D010618:Phenanthrolines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D015032:Zinc / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D002614:Chelating Agents; D010618:Phenanthrolines; D011994:Recombinant Proteins; D004492:Edetic Acid; D018826:CD13 Antigens; D015032:Zinc; C025205:1,10-phenanthroline