title: Inhibition of mitochondrial F1-ATPase activity by binding of (2-azido-) ADP to a slowly exchangeable non-catalytic nucleotide binding site.
abstract: F1-ATPase was treated so that it contained three tightly bound nucleotides per molecule. One of these was bound at a catalytic site and was rapidly exchangeable, the two remaining nucleotides were nonexchangeable. Incubation of this preparation with ADP in the presence of Mg2+ results in 40-45% inhibition of the ATPase activity. With 2-azido-ADP instead of ADP, the ligand was covalently bound to F1 by illumination, in the presence or absence of turnover of the enzyme, and the site of binding was determined. In this way, one site could be identified, which induces the inhibition. The attachment of the covalently bound 2-nitreno-ADP is at Tyr-368 of a beta-subunit, characterized in the literature as a non-catalytic site. A second, non-catalytic site also binds 2-azido-ADP, but this binding is partially reversed by the addition of ATP and does not cause further inhibition of the ATPase activity. It is concluded that the slowly exchangeable non-catalytic site is the site of inhibition by ADP.
authors.affiliation: E.C. Slater Institute for Biochemical Research, University of Amsterdam, Netherlands.
authors.forename: C M
authors.initials: CM
authors.lastname: Edel
authors.forename: A F
authors.initials: AF
authors.lastname: Hartog
authors.forename: J A
authors.initials: JA
authors.lastname: Berden
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004586:Electrophoresis; D008274:Magnesium / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*
chemical_list: D001386:Azides; D002413:Cations, Divalent; D009711:Nucleotides; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium