title: Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase.
abstract: Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.
mesh_terms: D025181:ATP Synthetase Complexes; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010101:Oxygen Consumption; D010710:Phosphates / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D013997:Time Factors
chemical_list: D009097:Multienzyme Complexes; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D025181:ATP Synthetase Complexes