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J. Biol. Chem., Vol. 279, Issue 37, 38861-38870, September 10, 2004

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Mechanistic Analysis of the Mitotic Kinesin Eg5^*

Jared C. Cochran, Christopher A. Sontag, Zoltan Maliga¶, Tarun M. Kapoor||, John J. Correia, and Susan P. Gilbert**

From the Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, the Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216, the ^¶Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, and the ^||Laboratory of Chemistry and Cell Biology, Rockefeller University, New York, New York 10021

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k_cat = 5.5 s^-1, K_1/2,Mt = 0.7 µM, and K_m,ATP = 25 µM; and for Eg5-437: k_cat = 2.9 s^-1, K_1/2,Mt = 4.5 µM, and K_m,ATP = 19 µM. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 µM^-1s^-1, followed immediately by ATP hydrolysis at 15 s^-1. ATP-dependent Mt·Eg5 dissociation was relatively slow and rate-limiting at 8 s^-1 with mantADP release at 40 s^-1. Surprisingly, Eg5-367 binds microtubules more effectively (11 µM^-1s^-1) than Eg5-437 (0.7 µM^-1s^-1), consistent with the steady-state K_1/2,Mt and the mantADP release K_1/2,Mt. These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.

Received for publication, April 15, 2004 , and in revised form, June 21, 2004.

^* This work was supported by Grant GM54141 from the NIGMS, National Institutes of Health (NIH) and through a Career Development Award K02-AR47841 from NIAMS, NIH, Department of Health and Human Services (to S. P. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 412-624-5842; Fax: 412-624-4759; E-mail: spg1{at}pitt.edu.

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