Abstract
The binding change model for the F(1)-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of approximately 40 piconewtons (pN).nm, and mechanical work done in a step of approximately 80 pN.nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was -80 pN.nm/molecule, indicating approximately 100% efficiency. Reconstituted F(o)F(1)-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F(1) very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.
Highlights
The FoF1 ATP synthase is an enzyme that synthesizes ATP from ADP and inorganic phosphate (Pi) using proton flow across a membrane [1]
We have shown that the purine ring of a nucleotide is indispensable for ␥ rotation and for proton pumping in the FoF1-ATPase
Our results are consistent with the report by Perlin et al [18], where GTP and ITP were shown to drive proton pumping in the FoF1-ATPase, and support the idea that nucleotide hydrolysis is coupled to proton pumping through mechanical rotation of the ␥ subunit
Summary
¶ Present address: Dept. of Structural Biology, Free University of Amsterdam, De Boelelaan 1087, 1081, Amsterdam, Netherlands. Single-molecule imaging of F1, in particular, has revealed that the ␥ subunit rotates in a unique direction consistent with the crystal structure, that ␥ makes discrete 120° steps, and that the energy conversion efficiency of the F1 motor driven by ATP hydrolysis can reach ϳ100% [9, 10]. In the reverse reaction of ATP hydrolysis, the free energy difference between the stronger ATP binding and weaker ADP/Pi binding drives the rotation of ␥. Boyer proposes that these binding changes occur sequentially on the three catalytic sites, synchronously with the ␥ rotation. In the crystal structure of F1 [5], the three  subunits carried a different nucleotide, an analog of ATP, ADP, and none, in support of the sequential binding change mechanism. Cleotides support F1 rotation, suggesting that the interaction between the catalytic site and the additional ring in purines is critical to the proper operation of this molecular machine
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