Section 69
Chapter 68,637

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

Cross, R.L.; Nalin, C.M.

Journal of Biological Chemistry 257(6): 2874-2881


ISSN/ISBN: 0021-9258
PMID: 6460765
Accession: 068636839

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5'-Adenylyl-.beta.,.gamma.-imidodiphosphate (AMP-PNP), a nonhydrolyzable analog of ATP, was 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 MgCl2 reveal 3 AMP-PNP binding sites on native F1, including 1 high affinity site, Kd = 18 nM, and 2 lower affinity sites, K.delta. = 1.0 .mu.M. An inhibition constant of 14 nM is obtained for AMP-PNP inhibition of ATP hydrolysis. Modification of essential amino acid residues of F1 with pyrixodal 5'-phosphate or phenylglyoxal results in the loss of 1 AMP-PNP binding site, while modification of essential carboxyl residues or the binding of efrapeptin results in the loss of 2 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 14C-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 retained 3 14C-labeled nucleotides. Subsequent incubation with [3H]AMP-PNP results in the binding of 3 mol of AMP-PNP/mol of F1 without displacement of any of the 14C-labeled nucleotide. There are 3 exchangeable nucleotide binding sites that are distinct from 3 noncatalytic sites, and there is a 3:3 stoichiometry for the .alpha. and .beta. subunits of mitochondrial F1. The exchangeable sites previously have 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.

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