Identification of the Nucleotide-Binding Site for ATP Synthesis and Hydrolysis in Mitochondrial Soluble F1-ATPase1

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Abstract
The binding of ADP to mitochondrial soluble F 1 -ATPase was studied by a membrane filtration method. One mol of F 1 , which contained about 1 mol of tightly bound adenine nucleotide, bound 4 mol of [α- 32 P]ADP at a saturating concentration of [α- 32 P]ADP. Two mol of the bound [α- 32 P]ADP was readily exchanged with medium nonradioactive ADP while the remaining 2 mol of the bound [α- 32 P]ADP was hardly exchanged (tightly bound [α- 32 P]ADP). F 1 catalyzed the synthesis of [α- 32 P]ATP from the medium [α- 32 P]ADP and P 1 . However, when exchangeably bound [α- 32 P]ADP was replaced by nonradioactive ADP, no [α- 32 P]ATP formation was observed. Furthermore, tightly bound [α- 32 P]ADP was not released from F 1 during ATP hydrolysis catalyzed by the enzyme. These results indicate that both ATP synthesis and hydrolysis catalyzed by F 1 occur at the exchangeable binding site and not at the tight binding site on the enzyme.