Uni-site catalysis in thylakoids. The influence of membrane energization on ATP hydrolysis and ATP-P_i exchange

ATP-hydrolysis was measured with thylakoid membranes during continuous illumination. The concentrations of free and enzyme-bound ATP, ADP and P_i were measured using either cold ATP, [γ-³²P]ATP or [¹⁴C]ATP. The concentration of free ATP was constant, free ADP and enzyme-bound ATP were below the detection limit. Nevertheless, [γ-³²P]ATP was bound, hydrolyzed and ³²P_i was released. The ADP was not released from the enzyme but cold Pi was bound from the medium, cold ATP was resynthesized and released. A quantitative analysis gave the following rate constants: ATP-binding k_ATP = 2·10⁵ M^-1s^-1, ADP-release: k_ADP <10^-2s^-1, P_i-release: k_Pi = 0.1 s^-1. These rate constants are considerably smaller than under deenergized conditions. The rate constant for the release of ATP can be estimated to be at least 0.2 s^-1 under energized conditions. Obviously, energization of the membrane, i.e. protonation of the enzyme leads mainly to a decrease of the rate of ATP-binding, to an increase of the rate of ATP release and to a decrease of the rate of ADP-release.