Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization

A truncated motor domain of the a subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH340) extends from the N terminus to amino acid 340. The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis of 0.029 +/- 0.002 s-1 in the absence of microtubules. The rate of release of bound ADP is 0.026 +/- 0.003 s-1. The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules. The rate of ATP hydrolysis is stimulated 3000-fold by addition of microtubules (MT) (kcat = 80 s-1; K(MT/0.5,ATPase) = 160 nM for half-saturation of the ATPase rate by microtubules at saturating ATP levels; K(MT/0.5,ATPase) = 43 micromolar for half-saturation of the ATPase rate by ATP at saturating microtubule levels). Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)triphosphate. One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules. In the presence of MgATP, K(MT/0.5,binding) for physical binding of DKH340 to microtubules is weaker than K(MT/0.5,ATPase) for stimulation of hydrolysis. These results are consistent with a model in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule. For this model, the kcat/K(MT/0.5,ATPase) ratio of 5 X 10(8) m-1 s-1 is at least as large as the bimolecular rate constant for association with microtubules, and this value approaches the diffusion controlled limit. Nucleotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgATP (t1/2 greater than or equal to 10 min), and thus it is likely to be in a conformational state which is not produced during steady state ATP hydrolysis.