DROSOPHILA KINESIN MINIMAL MOTOR DOMAIN EXPRESSED IN ESCHERICHIA-COLI- PURIFICATION AND KINETIC CHARACTERIZATION

A truncated motor domain of the alpha subunit of Drosophila kinesin wa s obtained by expression in Escherichia coli and purified to homogenei ty in the presence of MgATP. This domain (designated DKH340) extends f rom 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 appro ximate equality of the ADP release rate and the steady state ATPase ra te indicates that ADP release is the rate-limiting step in ATP hydroly sis in the absence of microtubules. The rate of ATP hydrolysis is stim ulated 3000 fold by addition of microtubules (MT) (k(cat) = 80 s(-1); K-0.5,ATPase(MT) = 160 nM for half-saturation of the ATPase rate by mi crotubules at saturating ATP levels; K-0.5,ATPase(MT) = 43 mu M for ha lf-saturation of the ATPase rate by ATP at saturating microtubule leve ls). Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)triphosphate. One DKH340 binds tightly per tubuli n heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/ microtubules. In the presence of MgA TP, K-0.5,Binding(MT) for physical binding of DKH340 to microtubules i s weaker than K-0.5,ATPase(MT) for stimulation of hydrolysis. These re - sults 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-0.5,ATPase(MT) ratio of 5 x 10(8) M(-1) s(-1) is at least as large as the bimolecular rate constant for association with microt ubules, and this value approaches the diffusion controlled limit. Nucl eotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgAT P (t(1/2) greater than or equal to 10 min), and thus it is likely to b e in a conformational state which is not produced during steady state ATP hydrolysis.