Basic Properties of Rotary Dynamics of the Molecular Motor Enterococcus hirae V1-ATPase

Background: The chemomechanical coupling scheme of the rotary motor V-1-ATPase is incompletely understood. Results:Enterococcus hirae V-1-ATPase (EhV(1)) showed 120 degrees steps of rotation without substeps, as commonly seen with F-1-ATPase. Conclusion: The basic properties of rotary dynamics of EhV(1) are similar to those of Thermus thermophilus V-1-ATPase. Significance: This study revealed the common properties of V-1-ATPases as rotary molecular motors, distinct from those of F-1-ATPases. V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V-1 moiety of Enterococcus hirae V-ATPase (EhV(1)) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV(1) using single-molecule analysis employing a load-free probe. EhV(1) rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (V-max) of 107 revolutions/s and a Michaelis constant (K-m) of 154 m at 26 degrees C. At all ATP concentrations tested, EhV(1) showed only three pauses separated by 120 degrees/turn, and no substeps were resolved, as was the case with Thermus thermophilus V-1-ATPase (TtV(1)). At 10 m ATP (?K-m), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 x 10(6) m(-1) s(-1). At 40 mm ATP (?K-m), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV(1). Our results identify the common properties of rotary catalysis of V-1-ATPases that are distinct from those of F-1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.