Effects of an ATP analogue, adenosine 5′-[α-thio]-triphosphate, on F1-ATPase rotary catalysis, torque generation, and inhibited intermediated formation

Yukawa, Ayako; Watanabe, Rikiya; Noji, Hiroyuki

doi:10.1016/j.bbrc.2015.01.146

Cited by 2 publications

(2 citation statements)

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“…We therefore reasoned that the use of an α-phosphate modified ATP prohibits the reaction with the luciferase but allows the reaction with ATPases. Indeed, commercially available ATPαS, in which the double-bonded oxygen on the α-phosphate is substituted for a sulfur, has been shown not to be a substrate for the luciferase in the deoxy form 35 , but is hydrolyzed by ATPases including the chloroplast F 1 F 0 ATP synthase 36 and the F 1 ATPase from Bacillus PS3 37 .…”

Section: Resultsmentioning

confidence: 99%

“…reported an apparent K m (MgATP) = 140 µM for E. coli F 1 F 0 ATP synthase 45 . Data for ATPαS with intact F 1 F 0 ATP synthase is missing, but in elegant single-molecule experiments following the rotation of the gamma subunit, Noji and coworkers have found an apparent K m (91 µM) with the purified F 1 ATPase from Bacillus PS3 lacking the regulatory epsilon subunit 37 . In comparison to published values, the Km values reported here were ~2-fold higher, which is likely due to different assay conditions (e.g.…”

Section: Discussionmentioning

confidence: 99%

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ATP synthesis at physiological nucleotide concentrations

Meyrat

Ballmoos

2019

Sci Rep

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%