Evidence that energization of the chloroplast ATP synthase favors ATP formation at the tight binding catalytic site and increases the affinity for ADP at another catalytic site.
Previous results have not established whether the attainment of a rapid photophosphorylation rate as ADP concentration is increased in the micromolar range (apparent Km = approximately 30 microM) results from the filling of a second or a third catalytic site. Measurements reported here show that the ATP synthase of chloroplast thylakoids, with 2-4 microM medium ADP present during steady-state photophosphorylation, has one catalytic site filled with tightly bound nucleotides, but other catalytic sites are largely empty. Thus, the rapid increase in the photophosphorylation rate with higher ADP concentrations results from the filling of a second catalytic site. Even with 30 microM added ADP in the dark, the binding of more than one ADP per synthase was not detectable. The sensitivity of the assay was such that the Kd for binding of ADP at a second catalytic site of the de-energized synthase is > 150 microM, considerably above the apparent Km for rapid photophosphorylation. This result can be explained by an increase in the affinity of a second catalytic site for ADP upon energization. Other experiments assessed the effect of ADP binding at a second catalytic site on the equilibrium between bound ATP and ADP and P(i) at the tight catalytic site. When the rate of photophosphorylation is limited by a low ADP concentration, about equal amounts of ATP and ADP are bound at one catalytic site on the synthase. In contrast, when the rate is limited by a low P(i) concentration with 100 microM ADP present, the equilibrium of bound reactants is shifted so that close to one ATP per synthase is present. This is as expected if the binding of ADP at a second catalytic site allows the protonmotive force to promote ATP formation from ADP and P(i) at a tight binding catalytic site. A scheme for the binding change mechanism incorporating these results is presented.
Zhou
J M
JM
Molecular Biology Institute, University of California, Los Angeles 90024-1570.
Boyer
P D
PD
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D004734:Energy Metabolism; D007425:Intracellular Membranes / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014675:Vegetables
D010710:Phosphates; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases