The Synthesis of Enzyme-bound ATP by Soluble Chloroplast Coupling

Purified CF1 (chloroplast coupling factor 1) synthesizes enzyme-bound ATP (CFltATP) from medium Pi. The reaction does not depend on medium ADP, indicating that the ADP substrate is tightly bound to CF1 (CFltADP). 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 CFl<ATP, showing that the ATP is never released from the enzyme. The addition of medium ATP, but not ADP, promotes the hydrolysis of CFl<ATP. The formation of CFl<ATP was analyzed in terms of a two-step reaction sequence in which Pi first binds to CFl<ADP which is then converted to CFl<ATP. Acid pH values were shown to increase the yield of CFl<ATP most significantly by promoting Pi binding. The equilibrium constant for the conversion of CFl<ADP=Pi to CFl tATP 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 HzP04species, which has been previously shown to be the form of phosphate that binds to beef heart Fl(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 CFl<ATP, if at the active site of photophosphorylation, means that protonmotive force does not directly promote he synthesis of the p-y phosphoryl bond of ATP during energy-driven ATP synthesis.