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

The binding of ADP to mitochondrial soluble F1-ATPase was studied by a membrane filtration method. One mol of F1, which contained about 1 mol of tightly bound adenine nucleotide, bound 4 mol of [alpha-32P]ADP at a saturating concentration of [alpha-32P]ADP. Two mol of the bound [alpha-32P]ADP was readily exchanged with medium nonradioactive ADP while the remaining 2 mol of the bound [alpha-32P] ADP was hardly exchanged (tightly bound [alpha-32P]ADP). F1 catalyzed the synthesis of [alpha-32P]ATP from the medium [alpha-32P]ADP and Pi. However, when exchangeably bound [alpha-32P]ADP was replaced by nonradioactive ADP, no [alpha-32P] ATP formation was observed. Furthermore, tightly bound [alpha-32P]ADP was not released from F1 during ATP hydrolysis catalyzed by the enzyme. These results indicate that both ATP synthesis and hydrolysis catalyzed by F1 occur at the exchangeable binding site and not at the tight binding site on the enzyme.