title: The rate-limiting step in microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule.
abstract: 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 MTs. 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 release and increased the rate of steady state ATP hydrolysis by almost 10,000-fold (kcat = approximately 45 s-1). The KMT0.5,ATPase value for saturation of the stimulation of the ATPase reaction by microtubules was 50 nM at 8 nM DKH392, but decreased at lower concentrations of DKH392. Physical binding of DKH392 to microtubules in the presence of 1 mM MgATP paralleled saturation of the stimulation of the ATPase activity by microtubules indicating that the rate-limiting step in microtubule-stimulated ATP hydrolysis occurs while DKH392 is bound to the microtubule. These results suggest that microtubule-stimulated ATP hydrolysis by DKH392 may be processive with the hydrolysis of multiple ATP molecules during each diffusional encounter of DKH392 with a microtubule.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
authors.forename: D D
authors.initials: DD
authors.lastname: Hackney
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D004330:Drosophila; D006868:Hydrolysis; D016547:Kinesins / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins