Nucleotide-binding sites on Escherichia coli F1-ATPase. Specificity of noncatalytic sites and inhibition at catalytic sites by MgADP.

Hyndman, David; Milgrom, Yakov M.; Bramhall, E A; Cross, Richard L.

doi:10.1016/s0021-9258(19)61988-5

Cited by 90 publications

(24 citation statements)

References 52 publications

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“…To test this, we measured inhibition by ventA in the presence of azide, a well-known inhibitor of F-ATPases from all sources. Azide inhibits F-ATPase by increasing the fraction of MgADP-inhibited enzyme 34 , 37 , 38 . Including 6 µM azide in the assay (Fig.…”

Section: Resultsmentioning

confidence: 99%

Complex effects of macrolide venturicidins on bacterial F-ATPases likely contribute to their action as antibiotic adjuvants

Milgrom

Duncan

2021

Sci Rep

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Bacterial energy metabolism is now recognized as a critical factor for the efficacy of antibiotics. The F-type ATPase/ATP synthase (FOF1) is a central player in cellular bioenergetics of bacteria and eukaryotes, and its potential as a selective antibiotic target has been confirmed by the success of bedaquiline in combatting multidrug-resistant tuberculosis. Venturicidin macrolides were initially identified for their antifungal properties and were found to specifically inhibit FOF1 of eukaryotes and bacteria. Venturicidins alone are not effective antibacterials but recently were found to have adjuvant activity, potentiating the efficacy of aminoglycoside antibiotics against several species of resistant bacteria. Here we discovered more complex effects of venturicidins on the ATPase activity of FOF1 in bacterial membranes from Escherichia coli and Pseudomonas aeruginosa. Our major finding is that higher concentrations of venturicidin induce time– and ATP–dependent decoupling of F1-ATPase activity from the venturicidin-inhibited, proton-transporting FO complex. This dysregulated ATPase activity is likely to be a key factor in the depletion of cellular ATP induced by venturicidins in prior studies with P. aeruginosa and Staphylococcus aureus. Further studies of how this functional decoupling occurs could guide development of new antibiotics and/or adjuvants that target the F-type ATPase/ATP synthase.

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Section: Resultsmentioning

confidence: 99%

Complex effects of macrolide venturicidins on bacterial F-ATPases likely contribute to their action as antibiotic adjuvants

Milgrom

Duncan

2021

Sci Rep

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“…Pauses are thought to be due to inhibitory Mg 2þ ADP in the active binding site. EF 1 shows [Mg 2þ ADP]-dependent inhibition of catalytic turnover in bulk assays [52], and [Mg 2þ ]-dependent activity [53] and inhibition [54,55] are rstb.royalsocietypublishing.org Phil Trans R Soc B 368: 20120023 thought to be due to stabilization of the inhibitory ADP molecule by Mg 2þ [56]. The propensity of TF 1 to lapse into the long pause was greatly increased by the presence of Mg 2þ ADP [17].…”

Section: Discussionmentioning

confidence: 99%

High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F ₁ -ATPase

Bilyard

Nakanishi‐Matsui

Steel

et al. 2013

Phil. Trans. R. Soc. B

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The rotary motor F 1 -ATPase from the thermophilic Bacillus PS3 (TF 1 ) is one of the best-studied of all molecular machines. F 1 -ATPase is the part of the enzyme F 1 F O -ATP synthase that is responsible for generating most of the ATP in living cells. Single-molecule experiments have provided a detailed understanding of how ATP hydrolysis and synthesis are coupled to internal rotation within the motor. In this work, we present evidence that mesophilic F 1 -ATPase from Escherichia coli (EF 1 ) is governed by the same mechanism as TF 1 under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF 1 molecules, we characterized the ATP-binding, catalytic and inhibited states of EF 1 . We also show that the ATP-binding and catalytic states are separated by 35±3°. At room temperature, chemical processes occur faster in EF 1 than in TF 1 , and we present a methodology to compensate for artefacts that occur when the enzymatic rates are comparable to the experimental temporal resolution. Furthermore, we show that the molecule-to-molecule variation observed at high ATP concentration in our single-molecule assays can be accounted for by variation in the orientation of the rotating markers.

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“…These observador, taken together with the finding reported here that the OL3/83 complex is relatively insensitive to stimulation by LDAO compared to the ^ complex, suggest that the y subunit must be present for retention of inhibitory MgADP in a catalytic site. Evidence has accumulated from several laboratories which suggests that the inactive complex containing inhibitory MgADP in a single catalytic site of Fj-ATPases is formed in a two-step process (Vasilyeva et al, 1982b;Murataliev & Milgrom, 1989;Murataliev, 1992;Hyndman et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

“…It has been postulated that a complex, designated FrADP•-Mg, which either is active (Vasilyeva et al, 1982a,b;Bulygin & Vinogradov, 1991) or is easily reactivated in the presence of MgATP (Milgrom & Murataliev, 1989;Murataliev, 1992;Murataliev et al, 1991;Hyndman et al, 1994), is formed initially which slowly isomerizes to a stable, inactive complex, designated F^-ADP-Mg. The complexes are proposed to be in an equilibrium which is shifted in the direction of the FrADP-Mg complex by activating anions such as bisulfate or bicarbonate which bind to unknown sites (Vasilyeva et al, 1982a) or binding ATP, or PP¡ in the case of MF), to noncatalytic sites (Jault & Allison, 1993;Kalashnikova et al, 1988;.…”

Section: Discussionmentioning

confidence: 99%

“…The complexes are proposed to be in an equilibrium which is shifted in the direction of the FrADP-Mg complex by activating anions such as bisulfate or bicarbonate which bind to unknown sites (Vasilyeva et al, 1982a) or binding ATP, or PP¡ in the case of MF), to noncatalytic sites (Jault & Allison, 1993;Kalashnikova et al, 1988;. In the presence of azide, the Fi*-ADP"Mg complex is converted to the more stable Fi^ADP-Mg-Na" complex which effectively decreases the concentration of the Fi*ADP-Mg complex (Vasilyeva et al, 1982b;Murataliev, 1992;Hyndman et al, 1994). Slow isomerization of the FvADP-Mg complex to the Fi*-ADP'Mg complex might represent movement of the y subunit to an abortive position, either in a random event during turnover or after loading a single catalytic site with MgADP when noncatalytic sites are not saturated with ATP.…”

Section: Discussionmentioning

confidence: 99%

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The .alpha.3.beta.3.gamma. complex of the F1-ATPase from thermophilic Bacillus PS3 containing the .alpha.D261N substitution fails to dissociate inhibitory MgADP from a catalytic site when ATP binds to noncatalytic sites

Jault

Matsui²,

Jault³

et al. 1995

Biochemistry

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ATP hydrolyses by the wild-type alpha 3 beta 3 gamma and mutant (alpha D261N)3 beta 3 gamma subcomplexes of the F1-ATPase from the thermophilic Bacillus PS3 have been compared. The wild-type complex hydrolyzes 50 microM ATP in three kinetic phases: a burst decelerates to an intermediate phase, which then gradually accelerates to a final rate. In contrast, the mutant complex hydrolyzes 50 microM or 2 mM ATP in two kinetic phases. The mutation abolishes acceleration from the intermediate phase to a faster final rate. Both the wild-type and mutant complexes hydrolyze ATP with a lag after loading a catalytic site with MgADP. The rate of the MgADP-loaded wild-type complex rapidly accelerates and approaches that observed for the wild-type apo-complex. The MgADP-loaded mutant complex hydrolyzes ATP with a more pronounced lag, and the gradually accelerating rate approaches the slow, final rate observed with the mutant apo-complex. Lauryl dimethylamide oxide (LDAO) stimulates hydrolysis of 2 mM ATP catalyzed by wild-type and mutant complexes 4- and 7.5-fold, respectively. The rate of release of [3H]ADP from the Mg[3H]ADP-loaded mutant complex during hydrolysis of 40 microM ATP is slower than observed with the wild-type complex. LDAO increases the rate of release of [3H]ADP from the preloaded wild-type and mutant complexes during hydrolysis of 40 microM ATP. Again, release is slower with the mutant complex. When the wild-type and mutant complexes are irradiated in the presence of 2-N3-[3H]ADP plus Mg2+ or 2-N3-[3H]ATP plus Mg2+ and azide, the same extent of labeling of noncatalytic sites is observed. Whereas ADP and ATP protect noncatalytic sites of the wild-type and mutant complexes about equally from labeling by 2-N3-[3H]ADP or 2-N3-[3H[ATP, respectively, AMP-PNP provides little protection of noncatalytic sites of the mutant complex. The results suggest that the substitution does not prevent binding of ADP or ATP to noncatalytic sites, but rather that it affects cross-talk between liganded noncatalytic sites and catalytic sites which is necessary to promote dissociation of inhibitory MgADP.

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Nucleotide-binding sites on Escherichia coli F1-ATPase. Specificity of noncatalytic sites and inhibition at catalytic sites by MgADP.

Cited by 90 publications

References 52 publications

Complex effects of macrolide venturicidins on bacterial F-ATPases likely contribute to their action as antibiotic adjuvants

Complex effects of macrolide venturicidins on bacterial F-ATPases likely contribute to their action as antibiotic adjuvants

High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F ₁ -ATPase

The .alpha.3.beta.3.gamma. complex of the F1-ATPase from thermophilic Bacillus PS3 containing the .alpha.D261N substitution fails to dissociate inhibitory MgADP from a catalytic site when ATP binds to noncatalytic sites

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Nucleotide-binding sites on Escherichia coli F1-ATPase. Specificity of noncatalytic sites and inhibition at catalytic sites by MgADP.

Cited by 90 publications

References 52 publications

Complex effects of macrolide venturicidins on bacterial F-ATPases likely contribute to their action as antibiotic adjuvants

Complex effects of macrolide venturicidins on bacterial F-ATPases likely contribute to their action as antibiotic adjuvants

High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F 1 -ATPase

The .alpha.3.beta.3.gamma. complex of the F1-ATPase from thermophilic Bacillus PS3 containing the .alpha.D261N substitution fails to dissociate inhibitory MgADP from a catalytic site when ATP binds to noncatalytic sites

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High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F ₁ -ATPase