THE RATE-LIMITING STEP IN MICROTUBULE-STIMULATED ATP HYDROLYSIS BY DIMERIC KINESIN HEAD DOMAINS OCCURS WHILE BOUND TO THE MICROTUBULE

DKH392 is a construct which contains the first 392 amino acids of the alpha-subunit of Drosophila kinesin and is dimeric in solution (Huang, T.-G., Suhan, J., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16502 -16507). The ATPase rate of DKH392 was 0.005 s(-1) in the absence of M Ts. One ADP bound tightly to each subunit and the release of this ADP was the rate-limiting step in ATP hydrolysis. Microtubules accelerated the rate of ADP re- lease and increased the rate of steady state ATP hydrolysis by almost 10,000-fold (k(cat) = similar to 45 s(-1)). The K -0.5,ATPase(MT) value for saturation of the stimulation of the ATPase reaction by microtubules was 50 nM at 8 nM DKH392, but decreased at lo wer concentrations of DKH392. Physical binding of DKH392 to microtubul es in the presence of 1 mm MgATP paralleled saturation of the stimulat ion of the ATPase activity by microtubules indicating that the rate li miting step in microtubule-stimulated ATP hydrolysis occurs while DKH3 92 is bound to the microtubule. These results suggest that microtubule -stimulated ATP hydrolysis by DKH392 may be processive with the hydrol ysis of multiple ATP molecules during each diffusional encounter of DK H392 with a microtubule.