Robustness of Rotary Catalysis Mechanism of F1-ATPase

Abstract

Motor proteins convert chemical energy of ATP hydrolysis into mechanical force and movement, which play various physiological roles, such as organelle transport, muscular contraction, and energy synthesis. Among them, F1-ATPase (F1), rotary motor protein, has a unique feature to achieve the extremely high chemo-mechanical coupling efficiency and reversibility. To understand the energy coupling mechanism of F1, extensive studies have been done for identifying the important interactions with ATP in the catalytic site. The interaction with phosphate moiety of ATP via three charged amino-acid residues (p-loop lysine, general base, and arginine-finger), which are well conserved in p-loop NTPases including motor proteins, is the most crucial for catalysis; e.g., upon the genetic depletion of these charged residues, the mutant F1s do not show detectable catalytic activity. On the other hand, the interaction with base moiety of ATP via phenylalanine residues is crucial for binding of ATP to the catalytic site; e.g., upon the substitution of base group to uracil, the binding affinity to F1 is extremely weakened. In the present study, we retested the competency of catalysis and force generation by using the charge-depleted mutants or uracil-substituted substrate (UTP) in the single-molecule rotation assay, which offers us to assess them with great sensitivity and preciseness. Surprisingly, all mutants showed the processive rotation with a constant rotary torque, even though the binding or hydrolysis rate of ATP was extremely slowed down by a factor of 10,000. Thus, the chemo-mechanical coupling mechanism of F1 is found to be prominently robust; the catalysis is extremely tightly coupled to the torque generation, which probably contributes to its high efficiency and reversibility.