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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.


Activation of TF1(ΔNC) by the addition of εR126A. The ATPase activity of TF1(ΔNC) was measured by the decrease of absorbance at 340 nm using the NADH-coupled ATP-regenerating system at 50 μM ATP. The reaction was initiated by the addition of TF1(ΔNC) lacking ε at the time indicated by the left arrowhead. εWT, εR126A (360 nM), or buffer (−ε), was added at the time indicated by the right arrowhead. Vertical and horizontal bars denote 0.01 absorbance and 200 s, respectively.
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f6-6_59: Activation of TF1(ΔNC) by the addition of εR126A. The ATPase activity of TF1(ΔNC) was measured by the decrease of absorbance at 340 nm using the NADH-coupled ATP-regenerating system at 50 μM ATP. The reaction was initiated by the addition of TF1(ΔNC) lacking ε at the time indicated by the left arrowhead. εWT, εR126A (360 nM), or buffer (−ε), was added at the time indicated by the right arrowhead. Vertical and horizontal bars denote 0.01 absorbance and 200 s, respectively.

Mentions: TF1 comprising the K175A/T176A double mutant of the α subunit, TF1(NC), lacks the nucleotide binding ability of the non-catalytic sites on α subunits and its ADP-Mg inhibition is known to be very strong15 (recovery from F1*ADP-Mg hardly occurs). To examine further the relationship between the ε inhibition and the ADP-Mg inhibition, the effect of the ε subunit on the ATPase activity of TF1(ΔNC) was examined. If the ε inhibition is through stabilization of the ADP-Mg inhibition, addition of ε should result in even stronger inhibition. The results are shown in Figure 6. TF1(ΔNC) showed a very low ATPase activity, as previously reported15. Although no effect on the ATPase activity was observed with εWT, when εR126A was added, recovery of the ATPase activity was observed, contrary to the above prediction (Fig. 6). Because the activation is specific to the mutant ε that is essentially deficient in ATP binding, it is likely that the extended-state conformation of the C-terminal domain is involved in TF1(ΔNC)’s escape from ADP-Mg inhibition. The extended-state conformation of the ε subunit is known to reduce the affinity of the catalytic sites of TF1 to ADP by 1 to 3 orders of magnitude20. The conformational change of the ε subunit in the ADP-Mg inhibited F1 (F1*ADP-Mg in Fig. 4) from the intermediate- to the extended-states may facilitate direct escape from the ADP-Mg inhibited state (F1*ADP-Mg) to the ε inhibited state (F1*ε) (a route not shown in Fig. 4), resulting in the recovery of the ATPase activity of TF1(ΔNC) though weak (Fig. 6).


Inhibition of thermophilic F 1 -ATPase by the ε subunit takes different path from the ADP-Mg inhibition
Activation of TF1(ΔNC) by the addition of εR126A. The ATPase activity of TF1(ΔNC) was measured by the decrease of absorbance at 340 nm using the NADH-coupled ATP-regenerating system at 50 μM ATP. The reaction was initiated by the addition of TF1(ΔNC) lacking ε at the time indicated by the left arrowhead. εWT, εR126A (360 nM), or buffer (−ε), was added at the time indicated by the right arrowhead. Vertical and horizontal bars denote 0.01 absorbance and 200 s, respectively.
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Related In: Results  -  Collection

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f6-6_59: Activation of TF1(ΔNC) by the addition of εR126A. The ATPase activity of TF1(ΔNC) was measured by the decrease of absorbance at 340 nm using the NADH-coupled ATP-regenerating system at 50 μM ATP. The reaction was initiated by the addition of TF1(ΔNC) lacking ε at the time indicated by the left arrowhead. εWT, εR126A (360 nM), or buffer (−ε), was added at the time indicated by the right arrowhead. Vertical and horizontal bars denote 0.01 absorbance and 200 s, respectively.
Mentions: TF1 comprising the K175A/T176A double mutant of the α subunit, TF1(NC), lacks the nucleotide binding ability of the non-catalytic sites on α subunits and its ADP-Mg inhibition is known to be very strong15 (recovery from F1*ADP-Mg hardly occurs). To examine further the relationship between the ε inhibition and the ADP-Mg inhibition, the effect of the ε subunit on the ATPase activity of TF1(ΔNC) was examined. If the ε inhibition is through stabilization of the ADP-Mg inhibition, addition of ε should result in even stronger inhibition. The results are shown in Figure 6. TF1(ΔNC) showed a very low ATPase activity, as previously reported15. Although no effect on the ATPase activity was observed with εWT, when εR126A was added, recovery of the ATPase activity was observed, contrary to the above prediction (Fig. 6). Because the activation is specific to the mutant ε that is essentially deficient in ATP binding, it is likely that the extended-state conformation of the C-terminal domain is involved in TF1(ΔNC)’s escape from ADP-Mg inhibition. The extended-state conformation of the ε subunit is known to reduce the affinity of the catalytic sites of TF1 to ADP by 1 to 3 orders of magnitude20. The conformational change of the ε subunit in the ADP-Mg inhibited F1 (F1*ADP-Mg in Fig. 4) from the intermediate- to the extended-states may facilitate direct escape from the ADP-Mg inhibited state (F1*ADP-Mg) to the ε inhibited state (F1*ε) (a route not shown in Fig. 4), resulting in the recovery of the ATPase activity of TF1(ΔNC) though weak (Fig. 6).

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.