ATP synthesis at physiological nucleotide concentrations

Abstract Synthesis of ATP by the F 1 F 0 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 F 1 F 0 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 P i , 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.