Phosphate release from myosin Va occurs after the initial powerstroke but before the secondary powerstroke associated...

Myosins transduce chemical energy into mechanical work, with key steps being the powerstroke and release of phosphate (P_i) from the active site. This process is still poorly understood, in part, because the order of these events remains controversial. We used two independent approaches to determine if P_i was released before or after the initial powerstroke with a single-headed myosin Va in a laser trap assay, at 100 μM ATP. First, we determined the effect of P_i, and resistive load, on the size of the primary powerstroke, secondary powerstroke (hitch) and the duration of the actomyosin binding event (t_on). At low resistive load, 30 mM P_i did not alter the powerstroke, hitch or t_on. However, at higher resistive loads P_i significantly (p < 0.05) reduced t_on and eliminated the hitch while the powerstroke was unaffected. The second approach utilized a myosin Va construct containing a mutation (S217A) that slows P_i-release from the active site. The powerstroke and the hitch were unaffected at every resistive load at 0 mM P_i, but the hitch and t_on were significantly reduced by 30 mM P_i at the highest resistive load. These observations suggest that the primary powerstroke occurs with P_i in active site, implying that P_i-release follows the powerstroke. The observation that P_i eliminates the hitch at high resistive load suggests that P_i-release, and therefore P_i-rebinding, occurs between the primary powerstroke and hitch. Thus, these results suggest the order of the key events in the energy transduction pathway of a prototypical molecular motor.