Kinetic Analysis Of Atp Synthesis Catalyzed By E. Coli Fof1 Atp Synthase Reconstituted Into Egg Yolk Liposomes: Evidence For Bi-Site Activation

Escherichia coli FOF1 ATP synthase was reconstituted into liposomes made of asolectin, soybean PC or egg yolk PC. The reconstitution system with egg yolk proteoliposomes gave the highest ATP synthase activity and ATP yield, and was used for analysis of the reaction characteristics. Under optimal conditions (ΔpH 3.4 at 37°C, ΔΨ = 109 mV, 10 μM valinomycin), the steady state rate of ATP synthesis reached 400 s-1. The dependency for Pi was hyperbolic over a range from 0.01 -5 mM. In contrast, variation of ADP concentration over a broad range (20 nM-2000 μM) revealed two apparent Km, one much less than 1 μM and the second at 11 μM. The apparent Km values for both substrates were independent of the membrane potential, ΔΨ. We propose that filling of two catalytic sites is sufficient and necessary for steady state ATP synthesis. Also, thiophosphate was found to be an uncompetitive inhibitor of ATP synthesis with respect to ADP, which implies an ordered substrate binding with ADP binding preceding phosphate binding. The data are in agreement with a reversible ATP synthesis-hydrolysis catalytic step with the ratio of the forward and reverse rate constants close to unity (Baylis Scanlon et al. J. Biol. Chem. 283, 26228-26240, 2008). In contrast to ATP hydrolysis where binding of Mg·ATP to the third catalytic site drives rotational catalysis, our results show that steady state ATP synthesis only requires binding of substrates to the second site.