Myosin X is a high duty ratio motor

Abstract

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 Kd of 1.6 μM attributed to the large dissociation rate constant (2.1 s-1). Vmax and KATPase of the actin-activated ATPase activity of M10IQ1 were 13.5 s-1 and 17.4 μM, 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 Vmax. The Pi-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 Km ATPase and Km motility. Our results suggest that myosin X is a high duty ratio motor. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.