Pathway of the microtubule-kinesin ATPase.
We have established pathway of the kinesin ATPase by direct measurement of each step in the pathway. Kinesin binds to microtubules with an 8-nm repeat and a stoichiometry of one kinesin monomer unit per tubulin dimer. Thus, the dimeric kinesin binds with both heads attached to the microtubule and on adjacent tubulin subunits. In the steady state, kinesin has a low ATPase activity that is limited by the rate of ADP release (< 0.01 s-1) in the absence of microtubules and is activated 2000-fold by the addition of microtubules to achieve a maximum rate of approximately 20 s-1. Transient-state kinetic analysis has provided direct measurement of individual steps of the reaction to define the pathway of the microtubule-kinesin ATPase. These studies establish that the rate-limiting step in the ATPase pathway is the release of the kinesin-product complex (K.ADP.P) from the microtubule following ATP hydrolysis. After phosphate release, the rebinding of kinesin-ADP to the microtubule is fast, accounting for the high activation of the ATPase at low microtubule concentration. This ATPase cycle explains the phenomenological differences between myosin and kinesin observed in motility assays. Kinesin remains associated with a microtubule through multiple rounds of hydrolysis, because it spends only a small fraction of its duty cycle in the dissociated state. The discussion of this paper will focus on the new data, their interpretation, and significance for mechanisms of force production. The ATPase coupling mechanism will be compared with dynein and myosin.
Johnson
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KA
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
Gilbert
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SP
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D004330:Drosophila / Q000378:metabolism; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D015394:Molecular Structure; D009068:Movement / Q000502:physiology; D009218:Myosins / Q000737:chemistry / Q000378:metabolism
D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins; D016547:Kinesins