Adenine nucleotide binding sites on beef heart F1-ATPase. Evidence for three exchangeable sites that are distinct from three noncatalytic sites.

Abstract

5'-Adenylyl-P,y-imidodiphosphate (AMP-PNP), a nonhydrolyzable analog of ATP, has been used to probe the exchangeable nucleotide binding sites on beef heart mitochondrial F1-ATPase.Equilibrium binding measurements at pH 8.0 in the presence of excess MgClz reveal three AMP-PNP binding sites on native F,, including one high affinity site, Kd = 18 n ~, and two lower affinity sites, Kd = 1.0 PM.An inhibition constant of 14 n~ is obtained for AMP-PNP inhibition of ATP hydrolysis.Modification of essential amino acid residues of F1 with pyridoxal 5'-phosphate or phenylglyoxal results in the loss of one AMP-PNP binding site, while modification of essential carboxyl residues or the binding of efrapeptin results in the loss of two sites.In contrast, neither 4-chloro-7-nitrobenzofurazan modification nor aurovertin binding affect the stoichiometry of AMP-PNP binding sites on F1.F1, treated to remove nucleotides bound at noncatalytic sites, was reconstituted with l4C-labeled nucleotide.Following a short incubation with nonradioactive ATP under conditions that promote release of 14C label from exchangeable, but not from noncatalytic sites, the enzyme was found to retain three 14C-labeled nucleotides.Subsequent incubation with 13H]AMP-PNP results in the binding of 3 mol of AMP-PNP/mol of F1 without displacement of any of the 14C-labeled nucleotide.The results demonstrate the presence of three exchangeable nucleotide binding sites that are distinct from three noncatalytic sites, and support a 3:3 stoichiometry for the a and P subunits of mitochondrial FI.The exchangeable sites have previously been suggested to participate in catalysis and results presented here are consistent with such a role.A comparison of the Kd and Ki values obtained for AMP-PNP shows that the binding of a single mol of AMP-PNP/mol of F1 is sufficient to inhibit catalysis.Combined with evidence for cooperativity in binding nucleotides at the exchangeable sites, these results provide additional support for strong cooperative interactions between the multiple copies of the catalytic subunit present on F1.Catalytic sites for ATP synthesis by oxidative phosphorylation and photophosphorylation are present on the terminal