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

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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.


Comparison of relative ATPase activity and occupancy of the active state. The occupancy of the active state was the sum of the durations in the active state divided by the same sum plus the sum for the inactive state. The obtained occupancy for TF1(εR126A) was divided by that for TF1(−ε) and plotted against the ATP concentration (closed circles). ATPase activity of TF1(εR126A) was divided by that of TF1(−ε) and plotted against the ATP concentration (open squares).
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f5-6_59: Comparison of relative ATPase activity and occupancy of the active state. The occupancy of the active state was the sum of the durations in the active state divided by the same sum plus the sum for the inactive state. The obtained occupancy for TF1(εR126A) was divided by that for TF1(−ε) and plotted against the ATP concentration (closed circles). ATPase activity of TF1(εR126A) was divided by that of TF1(−ε) and plotted against the ATP concentration (open squares).

Mentions: The kinetic constants obtained by our analysis (Table 1) are not far from those expected from biochemical experiments12. The occupancy of the active-state ((sum of durations of the active states) / (sum of durations of the inactive and the active states)) was calculated for TF1(εR126A) and divided by that of TF1(−ε). This ratio had a dependence on the ATP concentration which was similar to the ATP dependence of the relative ATPase activity of TF1(εR126A) to TF1(−ε) (Fig. 5). The agreement shows that the results by the two methods are consistent. However, our values are different from those reported by Tsumuraya et al.19. According to their report, the ε subunit prolonged the duration of the inactive state by 6 fold (from 110 s to 670 s) and the active state was not affected significantly (from 300 to 330 s) at 200 nM ATP19. On the basis of the reported apparent second-order rate constant for activation12, the activation of the ε inhibited TF1(εWT) is estimated to take about 3 days with 200 nM ATP and should not have been observable at the ATP concentration. As Tsumuraya et al. pre-incubated TF1(εWT) in a high concentration of ATP, which was necessary to observe the rotation of TF1(εWT) at low concentrations of ATP, the ε subunit in their study may have retained ATP and what was observed may have been the differences in the ADP-Mg inhibition between TF1(−ε) and TF1(εWT with bound ATP) rather than those in the ε inhibition.


Inhibition of thermophilic F 1 -ATPase by the ε subunit takes different path from the ADP-Mg inhibition
Comparison of relative ATPase activity and occupancy of the active state. The occupancy of the active state was the sum of the durations in the active state divided by the same sum plus the sum for the inactive state. The obtained occupancy for TF1(εR126A) was divided by that for TF1(−ε) and plotted against the ATP concentration (closed circles). ATPase activity of TF1(εR126A) was divided by that of TF1(−ε) and plotted against the ATP concentration (open squares).
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Related In: Results  -  Collection

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f5-6_59: Comparison of relative ATPase activity and occupancy of the active state. The occupancy of the active state was the sum of the durations in the active state divided by the same sum plus the sum for the inactive state. The obtained occupancy for TF1(εR126A) was divided by that for TF1(−ε) and plotted against the ATP concentration (closed circles). ATPase activity of TF1(εR126A) was divided by that of TF1(−ε) and plotted against the ATP concentration (open squares).
Mentions: The kinetic constants obtained by our analysis (Table 1) are not far from those expected from biochemical experiments12. The occupancy of the active-state ((sum of durations of the active states) / (sum of durations of the inactive and the active states)) was calculated for TF1(εR126A) and divided by that of TF1(−ε). This ratio had a dependence on the ATP concentration which was similar to the ATP dependence of the relative ATPase activity of TF1(εR126A) to TF1(−ε) (Fig. 5). The agreement shows that the results by the two methods are consistent. However, our values are different from those reported by Tsumuraya et al.19. According to their report, the ε subunit prolonged the duration of the inactive state by 6 fold (from 110 s to 670 s) and the active state was not affected significantly (from 300 to 330 s) at 200 nM ATP19. On the basis of the reported apparent second-order rate constant for activation12, the activation of the ε inhibited TF1(εWT) is estimated to take about 3 days with 200 nM ATP and should not have been observable at the ATP concentration. As Tsumuraya et al. pre-incubated TF1(εWT) in a high concentration of ATP, which was necessary to observe the rotation of TF1(εWT) at low concentrations of ATP, the ε subunit in their study may have retained ATP and what was observed may have been the differences in the ADP-Mg inhibition between TF1(−ε) and TF1(εWT with bound ATP) rather than those in the ε 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.