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Inhibition of thermophilic F 1 -ATPase by the ε subunit takes different path from the ADP-Mg inhibition

View Article: PubMed Central - PubMed

ABSTRACT

The F1-ATPase, the soluble part of FoF1-ATP synthase, is a rotary molecular motor consisting of α3β3γδε. The γ and ε subunits rotate relative to the α3β3δ sub-complex on ATP hydrolysis by the β subunit. The ε subunit is known as an endogenous inhibitor of the ATPase activity of the F1-ATPase and is believed to function as a regulator of the ATP synthase. This inhibition by the ε subunit (ε inhibition) of F1-ATPase from thermophilic Bacillus PS3 was analyzed by single molecule measurements. By using a mutant ε subunit deficient in ATP binding, reversible transitions between active and inactive states were observed. Analysis of pause and rotation durations showed that the ε inhibition takes a different path from the ADP-Mg inhibition. Furthermore, the addition of the mutant ε subunit to the α3β3γ sub-complex was found to facilitate recovery of the ATPase activity from the ADP-Mg inhibition. Thus, it was concluded that these two inhibitions are essentially exclusive of each other.

No MeSH data available.


Time-course of the rotation of beads attached to TF1(−ε), TF1(εWT) and TF1(εR126A). Typical examples of time-course of bead rotation at 200 μM ATP with (A) TF1(−ε), (B) TF1(εWT) and (C) TF1(εR126A) are shown. Data with similar bead sizes are shown.
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f1-6_59: Time-course of the rotation of beads attached to TF1(−ε), TF1(εWT) and TF1(εR126A). Typical examples of time-course of bead rotation at 200 μM ATP with (A) TF1(−ε), (B) TF1(εWT) and (C) TF1(εR126A) are shown. Data with similar bead sizes are shown.

Mentions: It has been proposed that ATP binding to the ε subunit of TF1 stabilizes its folded-state conformation, with which TF1 is in the ATPase-active state13. As shown in Figure 1, TF1 with the wild-type ε subunit (TF1(εWT)) showed essentially the same rotational behavior as the TF1 lacking ε subunit (TF1(−ε)). Because the ATP binding to the wild-type ε subunit is very strong with a Kd in the order of μM at room temperature12,13, once the ATPase is activated, it does not return to the ε inhibited state, when the ATP concentration is higher than the Kd. Use of a mutant ε subunit (εR126A), which is essentially deficient in ATP binding with a Kd larger than 1 mM13, allowed observation of reversible transition of TF1 into the ε inhibited state even with high concentrations of ATP (200 μM). The overall rotational rate of the TF1(εR126A) was clearly slower (Fig. 1). In a previous study, it was only at extremely low concentrations of ATP that such transitions were observed with TF1(εWT)19. It should be noted that reversible transitions, such as observed here with the mutant εR126A, are physiologically relevant, because the ATP binding to the wild-type ε subunit is weak at 65°C12, a physiological temperature of the Bacillus PS3. The repetitive transitions between the active and the inactive states allowed us to evaluate the lifetimes of the states. The distributions of the durations of the inactive state (pauses longer than 1 s) and of the active state (the state in between successive inactive states) are shown in Figures 2 and 3, respectively. Their lifetimes were estimated by fitting each panel of the data with a function with one or two exponentials (Figs. 2 and 3 and Table 1). The lifetimes of the inactive state (τi1 and τi2) were essentially the same among TF1(−ε), TF1(εWT), and TF1(εR126A) for various concentrations of ATP (Fig. 2 and Table 1). The inactive state observed with TF1(−ε) was the ADP-Mg inhibition, because the ATPase lacked the ε subunit. The inactive state observed with TF1(εWT) must have been also the ADP-Mg inhibition, because εWT must have been in the folded-state conformation with bound ATP at the ATP concentrations employed.


Inhibition of thermophilic F 1 -ATPase by the ε subunit takes different path from the ADP-Mg inhibition
Time-course of the rotation of beads attached to TF1(−ε), TF1(εWT) and TF1(εR126A). Typical examples of time-course of bead rotation at 200 μM ATP with (A) TF1(−ε), (B) TF1(εWT) and (C) TF1(εR126A) are shown. Data with similar bead sizes are shown.
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Related In: Results  -  Collection

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f1-6_59: Time-course of the rotation of beads attached to TF1(−ε), TF1(εWT) and TF1(εR126A). Typical examples of time-course of bead rotation at 200 μM ATP with (A) TF1(−ε), (B) TF1(εWT) and (C) TF1(εR126A) are shown. Data with similar bead sizes are shown.
Mentions: It has been proposed that ATP binding to the ε subunit of TF1 stabilizes its folded-state conformation, with which TF1 is in the ATPase-active state13. As shown in Figure 1, TF1 with the wild-type ε subunit (TF1(εWT)) showed essentially the same rotational behavior as the TF1 lacking ε subunit (TF1(−ε)). Because the ATP binding to the wild-type ε subunit is very strong with a Kd in the order of μM at room temperature12,13, once the ATPase is activated, it does not return to the ε inhibited state, when the ATP concentration is higher than the Kd. Use of a mutant ε subunit (εR126A), which is essentially deficient in ATP binding with a Kd larger than 1 mM13, allowed observation of reversible transition of TF1 into the ε inhibited state even with high concentrations of ATP (200 μM). The overall rotational rate of the TF1(εR126A) was clearly slower (Fig. 1). In a previous study, it was only at extremely low concentrations of ATP that such transitions were observed with TF1(εWT)19. It should be noted that reversible transitions, such as observed here with the mutant εR126A, are physiologically relevant, because the ATP binding to the wild-type ε subunit is weak at 65°C12, a physiological temperature of the Bacillus PS3. The repetitive transitions between the active and the inactive states allowed us to evaluate the lifetimes of the states. The distributions of the durations of the inactive state (pauses longer than 1 s) and of the active state (the state in between successive inactive states) are shown in Figures 2 and 3, respectively. Their lifetimes were estimated by fitting each panel of the data with a function with one or two exponentials (Figs. 2 and 3 and Table 1). The lifetimes of the inactive state (τi1 and τi2) were essentially the same among TF1(−ε), TF1(εWT), and TF1(εR126A) for various concentrations of ATP (Fig. 2 and Table 1). The inactive state observed with TF1(−ε) was the ADP-Mg inhibition, because the ATPase lacked the ε subunit. The inactive state observed with TF1(εWT) must have been also the ADP-Mg inhibition, because εWT must have been in the folded-state conformation with bound ATP at the ATP concentrations employed.

View Article: PubMed Central - PubMed

ABSTRACT

The F1-ATPase, the soluble part of FoF1-ATP synthase, is a rotary molecular motor consisting of α3β3γδε. The γ and ε subunits rotate relative to the α3β3δ sub-complex on ATP hydrolysis by the β subunit. The ε subunit is known as an endogenous inhibitor of the ATPase activity of the F1-ATPase and is believed to function as a regulator of the ATP synthase. This inhibition by the ε subunit (ε inhibition) of F1-ATPase from thermophilic Bacillus PS3 was analyzed by single molecule measurements. By using a mutant ε subunit deficient in ATP binding, reversible transitions between active and inactive states were observed. Analysis of pause and rotation durations showed that the ε inhibition takes a different path from the ADP-Mg inhibition. Furthermore, the addition of the mutant ε subunit to the α3β3γ sub-complex was found to facilitate recovery of the ATPase activity from the ADP-Mg inhibition. Thus, it was concluded that these two inhibitions are essentially exclusive of each other.

No MeSH data available.