Amino Acid Residues β139, β189, and β319 Modulate ADP-Inhibition in Escherichia coli H+-FOF1-ATP Synthase

Lapashina, Anna S.; Shugaeva, Tatiana E.; Березина, К.М.; Kholina, Tatiana D.; Feniouk, Boris A.

doi:10.1134/s0006297919040084

Cited by 5 publications

(3 citation statements)

References 24 publications

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“…MgADP also inhibits ATP hydrolysis in the holoenzyme F o F 1 -ATP synthase in inverted native membrane vesicles [41,42]. At an ADP/ATP ratio of 250 µM/750 µM, the ATP hydrolysis rate drops to ~40%, and at 600 µM/400 µM, the remaining ATP hydrolysis rate is only 10%.…”

Section: Discussionmentioning

confidence: 99%

Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping FoF1-ATP Synthase Trapped in Solution

Pérez

Heitkamp

Börsch

2023

IJMS

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FoF1-ATP synthases in mitochondria, in chloroplasts, and in most bacteria are proton-driven membrane enzymes that supply the cells with ATP made from ADP and phosphate. Different control mechanisms exist to monitor and prevent the enzymes’ reverse chemical reaction of fast wasteful ATP hydrolysis, including mechanical or redox-based blockade of catalysis and ADP inhibition. In general, product inhibition is expected to slow down the mean catalytic turnover. Biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work more slowly at a decreasing substrate/product ratio, or an increasing number of individual enzymes could be completely blocked. Here, we examined the effect of increasing amounts of ADP on ATP hydrolysis of single Escherichia coli FoF1-ATP synthases in liposomes. We observed the individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Förster resonance energy transfer (smFRET). Observation times of single FRET-labeled FoF1-ATP synthases in solution were extended up to several seconds using a confocal anti-Brownian electrokinetic trap (ABEL trap). By counting active versus inhibited enzymes, we revealed that ADP inhibition did not decrease the catalytic turnover of all FoF1-ATP synthases equally. Instead, increasing ADP in the ADP/ATP mixture reduced the number of remaining active enzymes that operated at similar catalytic rates for varying substrate/product ratios.

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Section: Discussionmentioning

confidence: 99%

Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping FoF1-ATP Synthase Trapped in Solution

Pérez

Heitkamp

Börsch

2023

IJMS

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“…According to this model, an increasing MgADP fraction in a series of ADP/ATP mixtures should not affect the maximum ATP hydrolysis rate vmax at high ATP concentrations but should shift the Michaelis-Menten constant KM for the half-maximal ATP hydrolysis rate to much higher ATP concentrations. MgADP inhibits ATP hydrolysis also in the holoenzyme FoF1-ATP synthase in inverted native membrane vesicles [41,42]. At an ADP/ATP ratio of 250 M/750 M, the ATP hydrolysis rate has dropped to ~40%, and at 600 M/400 M the remaining ATP hydrolysis rate is only 10%.…”

Section: Discussionmentioning

confidence: 99%

Mechanism of ADP-inhibited ATP hydrolysis in single proton-pumping F_oF₁-ATP synthase trapped in solution

Pérez

Heitkamp

Börsch

2023

Preprint

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FoF1-ATP synthases in mitochondria, in chloroplasts and in most bacteria are the proton-driven membrane enzymes supplying the cells with ATP made from ADP and phosphate. To monitor and prevent the reverse chemical reaction of fast wasteful ATP hydrolysis by the enzymes, different control mechanisms exist including mechanical or redox-based blockade of catalysis and ADP inhibition. In general product inhibition is expected to slow down the mean catalytic turnover. However, biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work slower at a decreasing substrate/product ratio, or more and more individual enzymes are blocked completely. Here, we examined how increasing amounts of ADP affected ATP hydrolysis of single Escherichia coli FoF1-ATP synthases in liposomes. We observed individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Forster resonance energy transfer (smFRET). Observation times of single FRET-labeled FoF1-ATP synthase in solution were increased up to seconds using a confocal Anti-Brownian electrokinetic trap (ABEL trap). By counting active versus inhibited enzymes we revealed that ADP inhibition did not decrease the catalytic turnover of all FoF1-ATP synthases equally. Instead, increasing ADP in the ADP/ATP mixture reduced the number of the remaining active enzymes which were operating at similar catalytic rates for varying substrate/product ratios.

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“…Subbacterial inverted membrane particles (SBP) from Escherichia coli strain BW25113 were provided by Anna Lapashina (Belozersky Institute, Moscow State University). The particles were prepared as described in [23]. Briefly, after growth cells were washed once with buffer containing 10 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, and 10% glycerol, resuspended in 25-30 ml of the same buffer, and disrupted by two consecutive passages through a French cell press (SLM Aminco, USA) at 1000 PSI.…”

Section: Escherichia Colimentioning

confidence: 99%

The mitochondria-targeted derivative of the classical uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone is an effective mitochondrial recoupler

et al. 2020

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The synthesis of a mitochondria-targeted derivative of the classical mitochondrial uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) by alkoxy substitution of CCCP with n-decyl(triphenyl)phosphonium cation yielded mitoCCCP, which was able to inhibit the uncoupling action of CCCP, tyrphostin A9 and niclosamide on rat liver mitochondria, but not that of 2,4-dinitrophenol, at a concentration of 1–2 μM. MitoCCCP did not uncouple mitochondria by itself at these concentrations, although it exhibited uncoupling action at tens of micromolar concentrations. Thus, mitoCCCP appeared to be a more effective mitochondrial recoupler than 6-ketocholestanol. Both mitoCCCP and 6-ketocholestanol did not inhibit the protonophoric activity of CCCP in artificial bilayer lipid membranes, which might compromise the simple proton-shuttling mechanism of the uncoupling activity on mitochondria.

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Amino Acid Residues β139, β189, and β319 Modulate ADP-Inhibition in Escherichia coli H+-FOF1-ATP Synthase

Cited by 5 publications

References 24 publications

Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping FoF1-ATP Synthase Trapped in Solution

Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping FoF1-ATP Synthase Trapped in Solution

Mechanism of ADP-inhibited ATP hydrolysis in single proton-pumping F_oF₁-ATP synthase trapped in solution

The mitochondria-targeted derivative of the classical uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone is an effective mitochondrial recoupler

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Amino Acid Residues β139, β189, and β319 Modulate ADP-Inhibition in Escherichia coli H+-FOF1-ATP Synthase

Cited by 5 publications

References 24 publications

Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping FoF1-ATP Synthase Trapped in Solution

Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping FoF1-ATP Synthase Trapped in Solution

Mechanism of ADP-inhibited ATP hydrolysis in single proton-pumping FoF1-ATP synthase trapped in solution

The mitochondria-targeted derivative of the classical uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone is an effective mitochondrial recoupler

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Mechanism of ADP-inhibited ATP hydrolysis in single proton-pumping F_oF₁-ATP synthase trapped in solution