Abstract
The epsilon subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the epsilon subunit lacked the C-terminal domain (FoF1epsilonDeltaC), and ATP synthesis driven by acid-base transition (DeltapH) and the K+-valinomycin diffusion potential (DeltaPsi) was compared in detail with that of the wild-type enzyme (FoF1epsilonWT). The turnover numbers (kcat) of FoF1epsilonWT were severalfold lower than those of FoF1epsilonDeltaC. FoF1epsilonWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1epsilonWT and FoF1epsilonDeltaC on various combinations of DeltapH and DeltaPsi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1epsilonWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1epsilonDeltaC. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.
Highlights
The inhibition of ATP hydrolysis by the C-terminal domain of ⑀ has been extensively studied, the effect on ATP synthesis is not fully understood
We generated an Escherichia coli FoF1 (EFoF1) mutant in which the ⑀ subunit lacked the C-terminal domain (FoF1⑀⌬C), and ATP synthesis driven by acid-base transition (⌬pH) and the K؉-valinomycin diffusion potential (⌬⌿) was compared in detail with that of the wild-type enzyme (FoF1⑀WT)
Solubilized FoF1⑀WT showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1⑀⌬C. These results suggest that the C-terminal domain of the ⑀ subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the ␥ subunit
Summary
Construction and Expression of the EFoF1 Mutant—A wildtype EFoF1 expression vector (pRA100) [32] was provided by Prof. The collected supernatant was injected into a HisTrap HP column (GE Healthcare) pre-equilibrated with 5 ml of buffer D (20 mM HEPES-KOH (pH 7.5), 500 mM NaCl, 5 mM MgCl2, 200 M ADP, 50 mM imidazole, 20% glycerol (v/v), 1ϫ protease inhibitor mixture, 5 mM PAB, 0.3% (w/v) C12E8, and 0.1% (w/v) E. coli total lipid (Avanti)). The pH of the or HEPES-NaOH (pH 7.5), 2.5 mM MgSO4, 0.1–50 mM phosphate, 2.2 mg of luciferin/luciferase mixture from ATP Bioluminescence Assay Kit CLS II (Roche Applied Science), 0.001–1 mM ADP, 36 nM valinomycin, and 1–300 mM KCl. The amount mixture of acidified proteoliposome suspension and base buffer (pHout) was directly measured using a glass electrode to determine ⌬pH (ϭ pHout Ϫ pHin).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
