Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization.
A truncated motor domain of the alpha 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; KMT0.5,ATPase = 160 nM for half-saturation of the ATPase rate by microtubules at saturating ATP levels; KMT0.5ATPase = 43 microns 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)t-riphosphate. 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, KMT0.5,Binding for physical binding of DKH340 to microtubules is weaker than KMT0.5,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/KMT0.5,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 > or = 10 min), and thus it is likely to be in a conformational state which is not produced during steady state ATP hydrolysis.
Huang
T G
TG
Department of Biological Sciences Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Hackney
D D
DD
D000244:Adenosine Diphosphate / Q000032:analysis / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000032:analysis; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D004330:Drosophila / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D016547:Kinesins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D009068:Movement; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins