Myosin X is a high duty ratio motor

Myosin X is expressed in a variety of cell types and plays a role in cargo movement and filopodia extension, but its mechanoenzymatic characteristics are not fully understood. Here we analyzed the kinetic mechanism of the ATP hydrolysis cycle of acto-myosin X using a single-headed construct (M10IQ1). Myosin X was unique for the weak strong actin binding state (AMD) with a K-d of 1.6 mu M attributed to the large dissociation rate constant (2.1 s(-1)). V-max and K-ATPase of the actin-activated ATPase activity of M10IQ1 were 13.5 s(-1) and 17.4 mu M, respectively. The ATP hydrolysis rate (> 100 s(-1)) and the phosphate release rate from acto-myosin X (> 100 s(-1)) were much faster than the entire ATPase cycle rate and, thus, not rate-limiting. The ADP off-rate from acto-myosin X was 23 s(-1), which was two times larger than the V-max. The P-i-burst size was low (0.46 mol/ mol), indicating that the equilibrium is significantly shifted toward the prehydrolysis intermediate. The steady-state ATPase rate can be explained by a combination of the unfavorable equilibrium constant of the hydrolysis step and the relatively slow ADP off-rate. The duty ratio calculated from our kinetic model, 0.6, was consistent with the duty ratio, 0.7, obtained from comparison of K-m ATPase and K-m motility. Our results suggest that myosin X is a high duty ratio motor.