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

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.

F-ATP-ase of Escherichia coli membranes: The ubiquitous MgADP-inhibited state and the inhibited state induced by the ε-subunit's C-terminal domain are mutually exclusive.

ATP synthases are important energy-coupling, rotary motor enzymes in all kingdoms of life. In all F-type ATP synthases, the central rotor of the catalytic F1 complex is composed of the γ subunit and the N-terminal domain (NTD) of the ε subunit. In the enzymes of diverse bacteria, the C-terminal domain of ε (εCTD) can undergo a dramatic conformational change to trap the enzyme in a transiently inactive state. This inhibitory mechanism is absent in the mitochondrial enzyme, so the εCTD could provide a means to selectively target ATP synthases of pathogenic bacteria for antibiotic development. For Escherichia coli and other bacterial model systems, it has been difficult to dissect the relationship between ε inhibition and a MgADP-inhibited state that is ubiquitous for FOF1 from bacteria and eukaryotes. A prior study with the isolated catalytic complex from E. coli, EcF1, showed that these two modes of inhibition are mutually exclusive, but it has long been known that interactions of F1 with the membrane-embedded FO complex modulate inhibition by the εCTD. Here, we study membranes containing EcFOF1 with wild-type ε, ε lacking the full εCTD, or ε with a small deletion at the C-terminus. By using compounds with distinct activating effects on F-ATP-ase activity, we confirm that εCTD inhibition and ubiquitous MgADP inhibition are mutually exclusive for membrane-bound E. coli F-ATP-ase. We determine that most of the enzyme complexes in wild-type membranes are in the ε-inhibited state (>50%) or in the MgADP-inhibited state (30%).

MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis.

Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K(m) values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.

ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F(1)-ATPase are affected by inorganic phosphate.

The effect of inorganic phosphate (P(i)) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F(1)-ATPase from beef heart mitochondria (ndMF(1)) has been investigated. It is shown for the first time that P(i) decreases the apparent rate constant of uni-site ATP binding by ndMF(1) 3-fold with the K(d) of 0.38+/-0.14mM. During uni-site ATP hydrolysis, P(i) also shifts equilibrium between bound ATP and ADP+P(i) in the direction of ATP synthesis with the K(d) of 0.17+/-0.03mM. However, 10mM P(i) does not significantly affect ATP binding during multi-site catalysis.

A bi-site mechanism for Escherichia coli F1-ATPase accounts for the observed positive catalytic cooperativity.

Nucleotide binding to nucleotide-depleted F(1)-ATPase from Escherichia coli (EcF(1)) during MgATP hydrolysis in the presence of excess epsilon subunit has been studied using a combination of centrifugal filtration and column-centrifugation methods. The results show that nucleotide-binding properties of catalytic sites on EcF(1) are affected by the state of occupancy of noncatalytic sites. The ATP-concentration dependence of catalytic-site occupancy during MgATP hydrolysis demonstrates that a bi-site mechanism is responsible for the positive catalytic cooperativity observed during multi-site catalysis by EcF(1). The results suggest that a bi-site mechanism is a general feature of F(1) catalysis.

Studies of nucleotide binding to the catalytic sites of Escherichia coli betaY331W-F1-ATPase using fluorescence quenching.

Most studies of nucleotide binding to catalytic sites of Escherichia coli betaY331W-F(1)-ATPase by the quenching of the betaY331W fluorescence have been conducted in the presence of approximately 20 mM sulfate. We find that, in the absence of sulfate, the nucleotide concentration dependence of fluorescence quenching induced by ADP, ATP, and MgADP is biphasic, revealing two classes of binding sites, each contributing about equally to the overall extent of quenching. For the high-affinity catalytic site, the K(d) values for MgADP, ADP, and ATP equal 10, 43, and 185 nM, respectively. For the second class of sites, the K(d) values for these ligands are approximately 1,000x larger at 8.1, 37, and 200 microM, respectively. The presence of sulfate or phosphate during assay results in a marked increase in the apparent K(d) values for the high-affinity catalytic site. The results show, contrary to earlier reports, that Mg(2+) is not required for expression of different affinities for a nucleotide by the three catalytic sites. In addition, they demonstrate that the fluorescence of the introduced tryptophans is nearly completely quenched when only two sites bind nucleotide. Binding of ADP to the third site with a K(d) near mM gives little fluorescence change. Many previous results of fluorescence quenching of introduced tryptophans appear to require reinterpretation. Our findings support a bi-site catalytic mechanism for F(1)-ATPase.

Rapid hydrolysis of ATP by mitochondrial F1-ATPase correlates with the filling of the second of three catalytic sites.

Strong positive catalytic cooperativity is a central feature of the binding change mechanism for F1-ATPases. However, a detail of the mechanism that remains controversial is whether the kinetic enhancement derived from using substrate-binding energy at one catalytic site to promote product release from another site occurs upon the filling of the second or third of three catalytic sites on F1. To address this question, we compare the ATP concentration dependence of the rate of ATP hydrolysis by F1 from beef heart mitochondria to the ATP concentration dependence of the level of occupancy of catalytic sites during steady-state catalysis as measured by a centrifuge filtration assay. A single Km(ATP) is observed at 77 +/- 6 microM. Analysis of the nucleotide-binding data shows that half-maximal occupancy of a second catalytic site occurs at 78 +/- 18 microM ATP. We conclude that ATP binding to a second catalytic site is sufficient to support rapid rates of catalysis.