Mechanism of inhibition by C-terminal α-helices of the ε subunit of Escherichia coli FoF1-ATP synthase

Abstract

The ε 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 α-helices of ε, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of ε 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 ε subunit lacked the C-terminal domain (FoF1εΔC), and ATP synthesis driven by acid-base transition (ΔpH) and the K+-valinomycin diffusion potential (ΔΨ) was compared in detail with that of the wild-type enzyme (FoF1εWT). The turnover numbers (kcat) of FoF1εWT were several fold lower than those of FoF1εΔC. FoF1εWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1εWT and FoF1εΔC on various combinations of ΔpH and ΔΨ was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of ε. Solubilized FoF1εWTalso showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1εΔC. These results suggest that the C-terminal domain of the ε subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.