The synthesis of enzyme-bound ATP by soluble chloroplast coupling factor 1.
Purified CF1 (chloroplast coupling factor 1) synthesizes enzyme-bound ATP (CF1 less than ATP) from medium Pi. The reaction does not depend on medium ADP, indicating that the ADP substrate is tightly bound to CF1 (CF1 less than ADP). At saturating [Pi] and at the pH optimum of 6.0, a yield of 0.25 mol of ATP/mol of CF1 was obtained. The addition of hexokinase and glucose does not reduce the yield of CF1 less than ATP, showing that the ATP is never released from the enzyme. The addition of medium ATP, but not ADP, promotes the hydrolysis of CF1 less than ATP. The formation of CF1 less than ATP was analyzed in terms of a two-step reaction sequence in which Pi first binds to CF1 less than ADP which is then converted to CF1 less than ATP. Acid pH values were shown to increase the yield of CF1 less than ATP most significantly by promoting Pi binding. The equilibrium constant for the conversion of CF1 less than ADP . Pi to CF1 less than ATP was the same (0.4 at pH 6.0 and 7.0. The data suggest that acid pH values stimulate Pi binding by increasing the concentration of the H2PO4- species, which has been previously shown to be the form of phosphate that binds to beef heart F1 (33). These studies provide another example of an enzyme that dramatically lowers the free energy difference between enzyme-bound reactants and products compared to that of the same reaction occurring free in solution. The formation of CF1 less than ATP, if at the active site of photophosphorylation, means that protonmotive force does not directly promote the synthesis of the beta-gamma phosphoryl bond of ATP during energy-driven ATP synthesis.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010944:Plants / Q000201:enzymology; D011485:Protein Binding; D006180:Proton-Translocating ATPases
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases