ATP synthesis at physiological nucleotide concentrations

Scientific Reports
Axel Meyrat, Christoph von Ballmoos

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.

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Citations

Aug 6, 2019·Chembiochem : a European Journal of Chemical Biology·Hendrik R SikkemaBert Poolman
Aug 8, 2020·International Journal of Molecular Sciences·Daniel Spari, Guido Beldi
Jul 23, 2019·Archives of Biochemistry and Biophysics·Samah RaheemZulfiqar Ahmad
Dec 16, 2020·Biochimica Et Biophysica Acta. Bioenergetics·Owen D JarmanJudy Hirst
Jul 14, 2021·The Journal of Physical Chemistry. B·Thomas Heitkamp, Michael Börsch
Oct 9, 2021·Nature Reviews. Molecular Cell Biology·Irene Vercellino, Leonid A Sazanov
Dec 14, 2021·Frontiers in Bioengineering and Biotechnology·Bingyu LiConghui You

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Methods Mentioned

BETA
Fluorescence
Assay

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