Source of Rapidly Labeled ATP Tightly Bound to Non‐catalytic Sites on the Chloroplast ATP Synthetase

Bound [³²P]ATP is found on deenergized, washed chloroplast thylakoids which were illuminated in the presence of ADP and [³²P]P_i. Tight binding of [³²P]ATP occurred both during and after energization. Different classes of bound [³²P]ATP were distinguished on the basis of their rates of formation, susceptibility to hexokinase and displacement by unlabeled ATP. The rates of formation and discharge of the rapidly labeled tighly bound ATP class were much lower than that of ATP formation. The level of this bound ATP saturates at lower concentrations of substrates than does the rate of phosphorylation. Unlabeled ATP, present in the reaction medium, displaces the rapidly labeled tightly bound ATP without affecting the rate of phosphorylation. We therefore conclude that the rapidly labeled bound ATP class does not fulfill the requirements expected for a catalytic intermediate and that the nucleotide tight binding site(s) on the ATP synthetase differ from the catalytic site(s) for ATP formation. Since the rapidly labeled tightly bound [³²P]ATP is not abolished by high concentrations of hexokinase, but is nevertheless displaced by exogenous ATP, we propose that tight binding of ATP to non‐catalytic sites occurs via a free species of newly synthesized ATP which diffuses slowly to the medium from a space accessible to ATP but not to hexokinase.