Purine but not pyrimidine nucleotides support rotation of F(1)-ATPase.
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.
CREST "Genetic Programming" Team 13, Teikyo University Biotechnology Research Center 3F, Nogawa 907, Miyamae-ku, Kawasaki 216-0001, Japan.
H
H
Noji
D
D
Bald
R
R
Yasuda
H
H
Itoh
M
M
Yoshida
K
K
Kinosita
D000255:Adenosine Triphosphate / Q000378:metabolism; D020134:Catalytic Domain; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011685:Purine Nucleotides / Q000378:metabolism*; D011742:Pyrimidine Nucleotides / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000378:metabolism
D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D007293:Inosine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D014544:Uridine Triphosphate