Abstract
Synthesis of ATP by the F1F0 ATP synthase in mitochondria and most bacteria is energized by the proton motive force (pmf) established and maintained by respiratory chain enzymes. Conversely, in the presence of ATP and in the absence of a pmf, the enzyme works as an ATP-driven proton pump. Here, we investigate how high concentrations of ATP affect the enzymatic activity of the F1F0 ATP synthase under high pmf conditions, which is the typical situation in mitochondria or growing bacteria. Using the ATP analogue adenosine 5′-O-(1-thiotriphosphate) (ATPαS), we have developed a modified luminescence-based assay to measure ATP synthesis in the presence of millimolar ATP concentrations, replacing an assay using radioactive nucleotides. In inverted membrane vesicles of E. coli, we found that under saturating pmf conditions, ATP synthesis was reduced to ~10% at 5 mM ATPαS. This reduction was reversed by ADP, but not Pi, indicating that the ATP/ADP ratio controls the ATP synthesis rate. Our data suggests that the ATP/ADP ratio ~30 in growing E. coli limits the ATP synthesis rate to ~20% of the maximal rate possible at the applied pmf and that the rate reduction occurs via product inhibition rather than an increased ATP hydrolysis rate.
Highlights
Life is energetically expensive and mostly paid in adenosine triphosphate (ATP)
The luciferase first catalyzes the binding of D-luciferin to the α-phosphate of ATP-Mg2+, forming PPi-Mg2+ and luciferyl-AMP, which is subsequently oxidized by oxygen to form AMP, CO2 and oxyluciferin in an excited state that emits a photon while returning to the ground state (Supplementary Fig. S1, for a review, see33,34)
We have introduced a novel, alternative, non-radioactive method to measure ATP synthesis in vitro under physiological nucleotide concentrations and high pmf, using ATPαS as a replacement for ATP
Summary
Life is energetically expensive and mostly paid in adenosine triphosphate (ATP). In all aerobic organisms, reducing equivalents from the oxidative breakdown of nutrients are converted to ATP in a process termed oxidative phosphorylation. A further role has been attributed to the ε subunit in the coupling of F1 reactions and F0 proton pumping[8,9,15,16,17] In addition to these mechanisms, all F1F0 ATP synthases show a decreased ATP hydrolysis rate in the presence of high ADP concentrations, a phenomenon known as the MgADP-inhibition of ATP hydrolysis[18,19]. (III) The third method is based on the conversion of produced ATP by coupled enzymatic reactions that can be detected by spectroscopy, e.g. addition of D-glucose in the presence of hexokinase and subsequent dephosphorylation by glucose-6-phosphate dehydrogenase in the presence of NAD(P) that is reduced to NAD(P)H and can be followed spectroscopically Similar to luciferase, this method cannot discriminate between present or freshly synthesized ATP and is less sensitive than the other methods. The applicability of the technique to more isolated systems was confirmed in ATP synthesis experiments in liposomes containing purified bo[3] oxidase and ATP synthase from E. coli
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