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 i ntimately correlated with the changes in the affinities of the three cataly tic sites for nucleotides. If so, subtle differences in the nucleotide stru cture may have pronounced effects on rotation. Here we show by single-molec ule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring i n purine is indispensable for proper operation of this molecular motor. Alt hough the three purine nucleotides were bound to the enzyme at different ra tes, all showed similar rotational characteristics: counterclockwise rotati on, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of similar to 40 piconewtons (pN)(.)n m, and mechanical work done in a step of similar to 80 pN(.)nm. These latte r characteristics are likely to be determined by the rotational mechanism b uilt 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 similar to 100% efficiency. Reconstitu ted F0F1-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F-1 very slowly hyd rolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.