Activation/inactivation and uni-site catalysis by the reconstituted ATP-synthase from chloroplasts

The proton-translocating ATP-synthase of chloroplasts CF₀F₁, was isolated and reconstituted into asolectin liposomes. CF₀F₁ can exist in at least four different states, oxidized or reduced, either inactive or active. These states are characterized by different kinetics of ADP binding: There is no binding of ADP to the inactive, oxidized state, the rate constant for ADP binding to the inactive, reduced state is 7 · 10² M^-1 · s^-1. ADP binding to the active, reduced state occurs under deenergized conditions with 10⁵ M^-1 · s^-1 and transforms the enzyme into the inactive, reduced state. Parallel to the ADP-dependent inactivation, the enzyme can also inactivate without ADP binding with a first-order rate constant of 7 · 10^-3 M^-1 · s^-1. With the active, reduced enzyme ATP-hydrolysis was measured under uni-site conditions as has been carried out with MF₁ (Grubmeyer, C., Cross, R.C. and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092-12100). The rate constant for ATP binding is 10⁶ M^-1 · s^-1, the 'equilibrium constant' on the enzyme EADPP_i EATP is 0.4. The rate constants for P_i release and ADP release are 0.2 s^-1 and 0.1 s^-1, respectively. This indicates that the enzyme carries out a complete turnover under uni-site conditions with rates much higher than that reported for MF₁.