Mechanisms by which reactions catalyzed by chloroplast coupling factor 1 are inhibited: ATP synthesis and ATP-water oxygen exchange

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Abstract
The ATP-H2O back-exchange reaction catalyzed by membrane-bound chloroplast (from spinach) coupling factor 1 (CF1) in the light is known to be extensive; each reacting ATP molecule nearly equilibrates its .gamma.-PO3 oxygens with H2O before it dissociates from the enzyme. Pi, Asi, ADP and GDP, alternate substrates of photophosphorylation, each inhibit the exchange reaction. At all concentrations of these substrate/inhibitor molecules tested, the high extent of exchange per molecule of ATP that reacts remains the same, while the number of ATP molecules experiencing exchange decreases. Thus, these inhibitors appear to act in a competitive-type manner, decreasing ATP turnover, as opposed to modulating the rate constants responsible for the partitioning of E.cntdot.ATP during the exchange reaction. This is consistent with the identity of CF1 catalytic sites for ATP-H2O back-exchange and ATP synthesis. Carbonyl cyanide m-chlorophenylhydrazone and NH4Cl (uncouplers of photophosphorylation) and phloridzin (an energy-transfer inhibitor) also lower the rate of ATP-H2O back-exchange; they too are found to act by decreasing the turnover of the ATP pool, not the extent of exchange per reacting ATP molecule. The extent of ATP-H2O forward oxygen exchange, which occurs during net ATP synthesis prior to product dissociation, is unaffected by uncouplers, whether catalyzed by native CF1 (ATPase latent) or the dithiothreitol/light-activated ATPase form. The mode of NH4Cl inhibition of the ATP synthesis reaction, therefore, is not through a change in the partitioning of the E.cntdot.ATP complex. This is important in view of the uncoupler-modulated partitioning of E.cntdot.ADP.cntdot.Pi observed during ATP hydrolysis in the same system [Sherman, P.A., and Wimmer, M.J. (1983) Eur. J. Biochem. 136,539-543]. Varying light intensity has no effect upon the extent of ATP-H2O forward oxygen exchange during ATP synthesis catalyzed by native CF1. However, once the enzyme has been ATPase activated, light intensity is found to modulate the partitioning of E.cntdot.ATP, in contrast to its lack of effect on the partitioning of E.cntdot.ADP.cntdot.Pi measured during ATP hydrolysis.