Kinetic evidence for multiple dynein ATPase sites.

We have examined the kinetics of ATP-induced dissociation of the microtubule-dynein complex at low ATP concentrations in the presence of vanadate, which inhibits the enzyme after the binding and hydrolysis of a single ATP per site (Shimizu, T., and Johnson, K. A. (1983) J. Biol. Chem. 258, 13833-13840). Four aspects of the dissociation reaction could not be explained by a model of dynein with a single ATP-sensitive microtubule binding site. First, titration of the light-scattering amplitude versus ATP concentration in the presence of vanadate gave Mr = 720,000/ATP binding site, indicating approximately 2.8 sites/2 million molecular weight particle. Second, the dissociation reaction was incomplete at concentrations of less than 2 microM ATP in the absence of vanadate, while the addition of vanadate led to complete dissociation at an increased rate. Third, the time course of dissociation induced by less than or equal to 1 microM ATP in the presence of vanadate was biphasic, with a small but distinct lag. Fourth, the ATP concentration dependence of the rate of dissociation in the absence of vanadate was concave upward at concentrations of ATP less than 5 microM, whereas the plot was linear in the presence of vanadate. These data suggest that dynein has three ATP-sensitive microtubule binding sites and each site must bind ATP for dynein to detach from the microtubule.