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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.


Schematic model of the relationship between ε inhibition and ADP-Mg inhibition. F1, F1*ε and F1*ADP-Mg represent F1 in active state, ε inhibited state and ADP-Mg inhibited state, respectively. The time constant for each path at 200 μM ATP is shown. The time constant for recovery from ε inhibition was taken from τi1 for TF1(εR126A). Values shown in parentheses, which represent time constants for the ADP-Mg inhibition, are from τa and τi1 for TF1(−ε). The time constant for the ε inhibition is calculated as 1/(1/τa(TF1(εR126A)) − 1/τa(TF1(−ε))) assuming that τa(TF1(εR126A)) contained contributions both from the ε inhibition and from the ADP-Mg inhibition (τa(TF1(−ε))).
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f4-6_59: Schematic model of the relationship between ε inhibition and ADP-Mg inhibition. F1, F1*ε and F1*ADP-Mg represent F1 in active state, ε inhibited state and ADP-Mg inhibited state, respectively. The time constant for each path at 200 μM ATP is shown. The time constant for recovery from ε inhibition was taken from τi1 for TF1(εR126A). Values shown in parentheses, which represent time constants for the ADP-Mg inhibition, are from τa and τi1 for TF1(−ε). The time constant for the ε inhibition is calculated as 1/(1/τa(TF1(εR126A)) − 1/τa(TF1(−ε))) assuming that τa(TF1(εR126A)) contained contributions both from the ε inhibition and from the ADP-Mg inhibition (τa(TF1(−ε))).

Mentions: Two exponentials were necessary to fit the data of the inactive state durations. The lifetimes were basically in agreement with those previously reported for the ADP-Mg inhibition16. The short lived inactive state (corresponding to τi2) was also observed as in a previous study. Although a slightly larger value was obtained for τi2 with TF1(εR126A) at 20 μM ATP (Fig. 2 and Table 1), we focus only on τi1 here, as in the previous study16. In contrast to the relative insensitivity of the lifetimes of the inactive state to the presence and the kind of the ε subunit or to the ATP concentration, the lifetime of the active state (τa) for TF1(εR126A) was different from that of TF1(−ε) or TF1(εWT) and depended on the ATP concentration (Fig. 3 and Table 1). It was shorter than that with TF1(−ε) or TF1(εWT) by over 10 fold at 20 μM ATP. As the ATP concentration was raised, the lifetime became longer, reaching the same level with TF1(−ε) or TF1(εWT) at 2 mM ATP. According to the previously proposed model that the ε subunit stabilizes the ADP-Mg inhibition18,19 and the ADP-Mg inhibition is prerequisite for the ε inhibition19, the lifetime of the active state should not be shortened by the presence of the ε subunit, while the lifetime of the inactive state could be affected. Thus, this has led us to consider an alternative scheme where the ε inhibition and the ADP-Mg inhibition are the parallel paths (Fig. 4). With this scheme, it can be estimated that most (ca. 86%) of the inhibited states of the TF1(εR126A) with 200 μM ATP must be by the ε. Distinction between the two inhibited states, apart from kinetic arguments, is currently not feasible, because the angular position of the γ subunit in the ε inhibition and that in the ADP-Mg inhibition are the same (data not shown), which supports a previous proposal17,19.


Inhibition of thermophilic F 1 -ATPase by the ε subunit takes different path from the ADP-Mg inhibition
Schematic model of the relationship between ε inhibition and ADP-Mg inhibition. F1, F1*ε and F1*ADP-Mg represent F1 in active state, ε inhibited state and ADP-Mg inhibited state, respectively. The time constant for each path at 200 μM ATP is shown. The time constant for recovery from ε inhibition was taken from τi1 for TF1(εR126A). Values shown in parentheses, which represent time constants for the ADP-Mg inhibition, are from τa and τi1 for TF1(−ε). The time constant for the ε inhibition is calculated as 1/(1/τa(TF1(εR126A)) − 1/τa(TF1(−ε))) assuming that τa(TF1(εR126A)) contained contributions both from the ε inhibition and from the ADP-Mg inhibition (τa(TF1(−ε))).
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Related In: Results  -  Collection

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f4-6_59: Schematic model of the relationship between ε inhibition and ADP-Mg inhibition. F1, F1*ε and F1*ADP-Mg represent F1 in active state, ε inhibited state and ADP-Mg inhibited state, respectively. The time constant for each path at 200 μM ATP is shown. The time constant for recovery from ε inhibition was taken from τi1 for TF1(εR126A). Values shown in parentheses, which represent time constants for the ADP-Mg inhibition, are from τa and τi1 for TF1(−ε). The time constant for the ε inhibition is calculated as 1/(1/τa(TF1(εR126A)) − 1/τa(TF1(−ε))) assuming that τa(TF1(εR126A)) contained contributions both from the ε inhibition and from the ADP-Mg inhibition (τa(TF1(−ε))).
Mentions: Two exponentials were necessary to fit the data of the inactive state durations. The lifetimes were basically in agreement with those previously reported for the ADP-Mg inhibition16. The short lived inactive state (corresponding to τi2) was also observed as in a previous study. Although a slightly larger value was obtained for τi2 with TF1(εR126A) at 20 μM ATP (Fig. 2 and Table 1), we focus only on τi1 here, as in the previous study16. In contrast to the relative insensitivity of the lifetimes of the inactive state to the presence and the kind of the ε subunit or to the ATP concentration, the lifetime of the active state (τa) for TF1(εR126A) was different from that of TF1(−ε) or TF1(εWT) and depended on the ATP concentration (Fig. 3 and Table 1). It was shorter than that with TF1(−ε) or TF1(εWT) by over 10 fold at 20 μM ATP. As the ATP concentration was raised, the lifetime became longer, reaching the same level with TF1(−ε) or TF1(εWT) at 2 mM ATP. According to the previously proposed model that the ε subunit stabilizes the ADP-Mg inhibition18,19 and the ADP-Mg inhibition is prerequisite for the ε inhibition19, the lifetime of the active state should not be shortened by the presence of the ε subunit, while the lifetime of the inactive state could be affected. Thus, this has led us to consider an alternative scheme where the ε inhibition and the ADP-Mg inhibition are the parallel paths (Fig. 4). With this scheme, it can be estimated that most (ca. 86%) of the inhibited states of the TF1(εR126A) with 200 μM ATP must be by the ε. Distinction between the two inhibited states, apart from kinetic arguments, is currently not feasible, because the angular position of the γ subunit in the ε inhibition and that in the ADP-Mg inhibition are the same (data not shown), which supports a previous proposal17,19.

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.