201. Mechanism of Inhibition by C-terminal α-Helices of the ϵ Subunit of Escherichia coil F0F1-ATP Synthase.
- Author
-
Iino, Ryota, Hasegawa, Rie, Tabata, Kazuhito V., and Noji, Hiroyuki
- Subjects
- *
ESCHERICHIA coli , *ADENOSINE triphosphatase , *HYDROLYSIS , *BIOSYNTHESIS , *PROTEOLYTIC enzymes - Abstract
TheE subunit of bacterial F[sub0]F[sub1]-ATP synthase (F[sub0]F[sub1]), 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 F[sub0]F[sub1] (EF[sub0]F[sub1]) mutant in which the &3x20AC; subunit lacked the C-terminal domain (F[sub0]F[sub1][supϵΔC]), and ATP synthesis driven by acid-base transition (ΔpH) and the K[sup+]-valinomycin diffusion potential (ΔΨ) was compared in detail with that of the wild-type enzyme (F[sub0]F[sub1][supϵWT]). The turnover numbers (kcat) of F[sub0]F[sub1][supϵWT] were severalfold lower than those of F[sub0]F[sub1][supϵΔC]. F[sub0]F[sub1][supϵWT] showed higher Michaelis constants (K,,). The dependence of the activities of F[sub0]F[sub1][supϵWT] and F[sub0]F[sub1][supϵΔC] 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 F[sub0]F[sub1][supϵWT] also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of F[sub0]F[sub1][supϵΔC]. These results suggest that the C-terminal domain of the ϵ subunit of EF[sub0]F[sub1] slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF