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 states is 7.10(2) M-1.s-1. ADP binding to the active, reduced state occurs under deenergized conditions with 10(5) 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 10(6) M-1.s-1, the 'equilibrium constant' on the enzyme EADPPi/EATP is 0.4. The rate constants for Pi release and ADP release are 0.2 s-1 and o.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.
Fromme
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Max-Volmer-Institut für Biophysikalische und Physikalische Chemie, Technische Universität Berlin (Germany).
Gräber
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010710:Phosphates; D011485:Protein Binding; D011489:Protein Denaturation; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D008081:Liposomes; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases