Inhibition and inactivation of the F1 adenosine triphosphatase from Bacillus PS3 by dequalinium and activation of the enzyme by lauryl dimethylamine oxide

of thermophilic F 1 -ATPase were prepared, and their catalytic properties were compared to know the role of ␦ and ⑀ subunits in catalysis. The presence of ␦ subunit in the complexes had slight inhibitory effect on the ATPase activity. The effect of ⑀ subunit was more profound. The (؊⑀) complexes, ␣ 3 ␤ 3 ␥ and ␣ 3 ␤ 3 ␥␦, initiated ATP hydrolysis without a lag. In contrast, the (؉⑀) complexes, ␣ 3 ␤ 3 ␥⑀ and ␣ 3 ␤ 3 ␥␦⑀, started hydrolysis of ATP (<700 M) with a lag phase that was gradually activated during catalytic turnover. As ATP concentration increased, the lag phase of the (؉⑀) complexes became shorter, and it was not observed above 1 mM ATP. Analysis of binding and hydrolysis of the ATP analog, 2 ,3 -O-(2,4,6-trinitrophenyl)-ATP, suggested that the (؉⑀) complexes bound substrate only slowly. Differing from Escherichia coli F 1 -ATPase, the activation of the (؉⑀) complexes from the lag phase was not due to dissociation of ⑀ subunit since the re-isolated activated complex retained ⑀ subunit. This indicates that there are two alternative forms of the (؉⑀) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.

Section: Fig 6 Re-isolation and Analysis Of Activated Subunit Complexmentioning

confidence: 44%

Thermophilic F1-ATPase Is Activated without Dissociation of an Endogenous Inhibitor, ε Subunit

Kato

Matsui²,

Tanaka

et al. 1997

“…2A compare hydrolysis of 2 mM ATP by the wild-type complex before and after incubation with ADP plus Mg 2ϩ . As observed for intact TF 1 (23,45), trace a illustrates that the wild-type ␣ 3 ␤ 3 ␥ complex hydrolyzes 2 mM ATP with a pronounced lag. The final rate of ATP hydrolysis in trace a is 18 mol/mg/min.…”

Section: Methodsmentioning

confidence: 67%

“…Three kinetic phases are observed when MF 1 , TF 1 , and the ␣ 3 ␤ 3 ␥ subcomplex of TF 1 hydrolyze low concentrations of ATP (8,22,23). An initial burst rapidly decelerates to a slow, intermediate rate, which, in turn, progressively accelerates to the final steady-state rate.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The α3β3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 with the βT165S Substitution Does Not Entrap Inhibitory MgADP in a Catalytic Site during Turnover

Jault

Dou

Grodsky

et al. 1996

Self Cite

The hydrolytic properties of the mutant ␣ 3 (␤T165S) 3 ␥ and wild-type ␣ 3 ␤ 3 ␥ subcomplexes of TF 1 have been compared. Whereas the wild-type complex hydrolyzes 50 M ATP in three kinetic phases, the mutant complex hydrolyzes 50 M ATP with a linear rate. After incubation with a slight excess of ADP in the presence of Mg 2؉ , the wild-type complex hydrolyzes 2 mM ATP with a long lag. In contrast, prior incubation of the mutant complex under these conditions does not affect the kinetics of ATP hydrolysis. The ATPase activity of the wild-type complex is stimulated 4-fold by 0.1% lauryl dimethylamine oxide, whereas this concentration of lauryl dimethylamine oxide inhibits the mutant complex by 25%. Compared with the wild-type complex, the activity of the mutant complex is much less sensitive to turnoverdependent inhibition by azide. This comparison suggests that the mutant complex does not entrap substantial inhibitory MgADP in a catalytic site during turnover, which is supported by the following observations. ATP hydrolysis catalyzed by the wild-type complex is progressively inhibited by increasing concentrations of Mg 2؉ in the assay medium, whereas the mutant complex is insensitive to increasing concentrations of Mg

“…S1 and S2). Lauryldodecylamine oxide has been thought to help F 1 not to lapse into ADP-Mg inhibition (23). The FRET change became faster in the presence of lauryldodecylamine oxide, while the time course of recovery of ATPase was almost unchanged 5 .…”

Section: Discussionmentioning

confidence: 99%

Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase

Iino

Murakami

Iizuka³

et al. 2005

It has been proposed that C-terminal two ␣-helices of the ⑀ subunit of F 1 -ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F 1 from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the ␥ subunit and C terminus of the ⑀ subunit, respectively. High FRET (extended form) of F 1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5-Adenyl-␤,␥-imidodiphosphate, ADP ؉ AlF 4 ؊ , ADP ؉ NaN 3 , and GTP also caused the retracted form, indicating that ATP binding to the catalytic ␤ subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the ⑀ subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65°C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated ⑀ subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65°C reached to 0.67 mM, a consistent value to assume that the ⑀ subunit acts as a sensor of ATP concentration in the cell.A rotary motor F 1 -ATPase (F 1 ) 2 is a water-soluble portion of F 0 F 1 -ATP synthase, which catalyzes ATP synthesis/hydrolysis coupled with a transmembrane proton translocation (1, 2). F 1 has a subunit structure of ␣ 3 ␤ 3 ␥␦⑀; ␣ and ␤ subunits have a non-catalytic and catalytic nucleotide binding sites, respectively; ␥ subunit rotates in the ␣ 3 ␤ 3 ring; ␦ subunit connects the ring to the stator part of F 0 ; and ⑀ subunit rotates together with ␥ subunit as a body. The ⑀ subunit (ϳ14 kDa) has a regulatory function and consists of N-terminal ␤-sandwitch and C-terminal two ␣-helices (3, 4).Previous structural studies of F 1 indicated two conformations of the ⑀ subunit with different arrangement of the two ␣-helices, that is, retracted folded-hairpin form and partly extended form (Fig. 1, A and B) (5-8). Cross-linking studies suggested the third conformation with fully extended ␣-helices 3 (Fig. 1C) (9). Biochemical data have indicated that the ⑀ subunit adopts the extended form in the absence of nucleotide or in the presence of ADP, in which ATPase activity is inhibited, and that ATP counteracts ADP by favoring the retracted form, which is a noninhibitory conformation (9). Thus, it appears that the regulatory function of the ⑀ subunit is dependent on the drastic conformational transition that is affected by nucleotide and other factors. However, previous studies have not provided kinetic information on how these dynamic conformational transitions occur in the enzyme at work. Fluorescence resonance energy transfer (FRET) is a powerful technique that enables us to probe conformational...