Timing of inorganic phosphate release modulates the catalytic activity of ATP-driven rotary motor protein

F 1 -ATPase is a rotary motor protein driven by ATP hydrolysis. The rotary motion of F 1 -ATPase is tightly coupled to catalysis, in which the catalytic sites strictly obey the reaction sequences at the resolution of elementary reaction steps. This fine coordination of the reaction scheme is thought to be important to achieve extremely high chemomechanical coupling efficiency and reversibility, which is the prominent feature of F 1 -ATPase among molecular motor proteins. In this study, we intentionally change the reaction scheme by using single-molecule manipulation, and we examine the resulting effect on the rotary motion of F 1 -ATPase. When the sequence of the products released, that is, ADP and inorganic phosphate, is switched, we find that F 1 frequently stops rotating for a long time, which corresponds to inactivation of catalysis. This inactive state presents MgADP inhibition, and thus, we find that an improper reaction sequence of F 1 -ATPase catalysis induces MgADP inhibition. The F1-ATPase is a motor protein which exhibits rotary motion as a result of catalytic hydrolysis of ATP. Here, the authors investigate how the sequence of this reaction influences molecular rotation, showing that premature product release can result in protein inactivation.