Pause and rotation of F(1)-ATPase during catalysis

F(1)-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120° rotation of the central γ subunit relative to the surrounding α(3)β(3) ring. Here, we show that the rotation of F(1)-ATPase spontaneously lapses into long (≈30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of F(1)-ATPase previously known as the ADP-Mg inhibited form in which F(1)-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the γ subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90° position.