title: Adenine nucleotide binding sites on beef heart F1-ATPase. Specificity of cooperative interactions between catalytic sites.
abstract: Cooperative interactions between nucleotide binding sites on beef heart mitochondrial F1-ATPase have been studied by measuring substrate-promoted release of 5'adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) from a single high affinity site. The site is initially loaded by incubating F1 with an equimolar amount of the nonhydrolyzable ATP analog. When unbound [3H]AMP-PNP is removed and the complex diluted to a concentration below the Kd, release of ligand shows an apparent absolute requirement for medium ADP. Release is biphasic with the extent of release during the initial rapid phase dependent on the concentration of medium ADP. Although phosphate alone has no effect, it enhances the rapid phase of ADP-promoted release over 2-fold with a half-maximal effect at 60 micrometers P1. The binding of efrapeptin (A23871) to the F1.AMP-PNP complex completely prevents ADP-promoted dissociation. Although AMP-PNP release also occurs in the presence of medium ATP, the F1.AMP-PNP complex does not dissociate if an ATP-regenerating system of sufficient capacity to prevent accumulation of medium ADP is added. Consistent with an inability of nucleoside triphosphate to promote release is the failure of medium, nonradioactive AMP-PNP to affect retention of the 3H-labeled ligand. The stability of F1.AMP-PNP complex in the absence of medium nucleotide and the highly specific ability of ADP plus P1 to promote rapid release of the ATP analog are interpreted as support for an ATP synthesis mechanism that requires substrate binding at one catalytic site for product release from an adjacent interacting site.
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010086:Oxidative Phosphorylation Coupling Factors / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D006180:Proton-Translocating ATPases; D011770:Pyruvate Kinase / Q000378:metabolism
chemical_list: D000227:Adenine Nucleotides; D010086:Oxidative Phosphorylation Coupling Factors; D010710:Phosphates; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases