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

Abstract

1. 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.2. 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.3. 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.With the discovery of ATP and ADP tightly bound to the ATP synthetase of mitochondria and chloroplasts, the question of their participation in the mechanism of the energytransduction process has been intensively investigated [I -41. Studies on binding, dissociation and phosphorylation of nucleotides associated with the tight binding site(s) of the chloroplast ATP synthetase [5 -71 have shown that these processes are energy dependent. The different kinetic properties of the exchange (i.e. release and binding) [8] and phosphorylation [9,10] of tightly bound ADP as compared to the phosphorylation of medium ADP indicate that the deenergized conformation of the ATP synthetase contains ADP bound to a site which is not the catalytic site for ATP formation. ATP has also been detected tightly bound to chloroplast thylakoids during [lo] and after energization [6,1 I]. Measurement of the rates of exchange of phosphate oxygens during net synthesis or hydrolysis of ATP and assessment of the amounts of reactants bound during catalysis led Boyer and coworkers [6,12,13] to formulate a mechanism for energy transduction in chloroplasts and mitochondria in which ATP, tightly bound to the ATP synthetase, participates as a catalytic intermediate in oxidative and photophosphorylation. In this mechanism, the release of ATP from one catalytic site is promoted by an energy-dependent conformational change in the ATP synthetase concomitant with the binding of ADP and Pi at an alternate catalytic site.The participation of a tightly bound ATP as a necessary catalytic intermediate is questionable since during steadystate phosphorylation of various nucleoside diphosphates Pi. From the rates of formation and release of the rapidly labeled tightly bound ATP and its exchangeability with medium ATP, we deduce that this ATP species is probably bound to non-catalytic sites on the ATP synthetase. MATERIALS AND METHODSChloroplast thylakoids were isolated from fresh market lettuce leaves by conventional procedures, washed once by resuspension in 0.4 M sucrose in 1 mM Tri...

An attempt to discriminate catalytic and regulatory proton binding sites in membrane-bound, thiol-reduced chloroplast ATPase

“…Another possibility is a direct transfer of ADP from the catalytic site to a regulatory site, without passing through the medium, which would increase the rate of deactivation of all states. The direct transfer of nucleotides from catalytic to regulatory sites, without equilibration with the medium, has been debated for more than ten years (Rosen et al, 1979;Aflalo & Shavit, 1982). This would lead to an hyperbolic relationship between V0 and k, maybe closer to the experimental situation (Figures 6 and 8).…”

Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

“…Likely, most of the light-promoted exchange of tightly bound nucleotides on thylakoid membranes that has been noted occurs with nucleotides bound at catalytic sites. Additional exploration of tight ATP binding on chloroplast thylakoids was undertaken in view of observations made in Shavit's laboratory (Bar-Zvi & Shavit, 1982; Aflalo & Shavit, 1982). They reported that after photophosphorylation with [32P]P¡ their washed thylakoid membrane preparations contained about 0.15-0.2 tightly bound ATP per Cp!…”

“…In contrast, ATP or 2-azido-ATP bound at the tight MgATP site remains as such. Thus, after photophosphorylation with [32P]P¡ under the conditions used in experiments reported from Shavit's laboratory (Bar-Zvi & Shavit, 1982; Aflalo & Shavit, 1982), some [32P]ATP binding at noncatalytic sites may occur, but, because of loss of the 32P-labeled -phosphoryl group, little or no 32P nucleotide binding at catalytic sites would be expected. Their interpretation that a tightly bound ATP at a catalytic site is not an intermediate in ATP synthesis, and that the major role of tightly bound ATP is in the modulation of the ATP synthase activities, does not seem appropriate in view of the findings reported here, as well as other evidence for a tightly bound ATP at catalytic sites as a transient catalytic intermediate.…”

Structure, function and regulation of chloroplast ATPase

“…Recent results, however, cast doubt on the significance of those experiments, since phosphorylation of other nucleoside diphosphates, like GDP, is not accompanied by rapid accumulation of the corresponding bound nucleoside triphosphates. Moreover, the rates of formation and discharge of the tightly bound ATP class are much lower than that of ATP formation (Aflalo and Shavit 1982). As shown below, tightly bound nucleotides play an important role in the regulation of the chloroplast ATPase.…”