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

ATP-hydrolysis was measured with thylakoid membranes during continuous illumination. The concentrations of free and enzyme-bound ATP, ADP and Pi were measured using either cold ATP, gamma-32P]ATP or 14C]ATP. The concentration of free ATP was constant, free ADP and enzyme-bound ATP were below the detection limit. Nevertheless, gamma-32P]ATP was bound, hydrolyzed and 32Pi 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 kATP = 2 . 10(5) M-1 s-1, ADP-release: kADP less than 10(-2)s-1, Pi-release: kPi = 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.