Steady state kinetics of ATP synthesis and hydrolysis catalyzed by reconstituted chloroplast coupling factor.

The steady state kinetics of ATP synthesis and hydrolysis catalyzed by the chloroplast dicyclohexylcarbodiimide-sensitive ATPase reconstituted into phospholipid vesicles were studied as a function of the transmembrane proton gradient. Bacteriorhodopsin also was incorporated into the vesicles so that a constant pH gradient could be maintained by continuous illumination of the liposomes. The dependence of the initial rates of ATP synthesis and hydrolysis on substrate concentrations is consistent with Michaelis-Menten kinetics, with enzyme, ADP, and Pi forming a ternary complex. The Michaelis constants for both synthesis and hydrolysis are essentially independent of the pH gradient, while the maximum velocities depend strongly on it. The equilibrium constant for hydrolysis was calculated from the steady state kinetic parameters, and the dependence of the equilibrium constant on the pH gradient indicates that 3 protons are transported per ATP synthesized or hydrolyzed. The dependence of the steady state kinetic parameters on the pH gradient can be described by a mechanism in which the binding of substrates occurs before the transport of protons and the transport of the 3 protons is sequential rather than concerted.