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J. Biol. Chem., Vol. 281, Issue 51, 39444-39454, December 22, 2006

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Dimeric Eg5 Maintains Processivity through Alternating-site Catalysis with Rate-limiting ATP Hydrolysis^*

From the Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Eg5/KSP is a homotetrameric, Kinesin-5 family member whose ability to cross-link microtubules has associated it with mitotic spindle assembly and dynamics for chromosome segregation. Transient-state kinetic methodologies have been used to dissect the mechanochemical cycle of a dimeric motor, Eg5-513, to better understand the cooperative interactions that modulate processive stepping. Microtubule association, ADP release, and ATP binding are all fast steps in the pathway. However, the acid-quench analysis of the kinetics of ATP hydrolysis with substrate in excess of motor was unable to resolve a burst of product formation during the first turnover event. In addition, the kinetics of P_i release and ATP-promoted microtubule-Eg5 dissociation were observed to be no faster than the rate of ATP hydrolysis. In combination the data suggest that dimeric Eg5 is the first kinesin motor identified to have a rate-limiting ATP hydrolysis step. Furthermore, several lines of evidence implicate alternating-site catalysis as the molecular mechanism underlying dimeric Eg5 processivity. Both mantATP binding and mantADP release transients are biphasic. Analysis of ATP hydrolysis through single turnover assays indicates a surprising substrate concentration dependence, where the observed rate is reduced by half when substrate concentration is sufficiently high to require both motor domains of the dimer to participate in the reaction.

Received for publication, August 22, 2006 , and in revised form, October 13, 2006.

^* This work was supported by the NIGMS, National Institutes of Health (NIH) Grant GM54141 and by 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Biological Sciences, University of Pittsburgh, 518 Langley Hall, Pittsburgh, PA 15260. Tel.: 412-624-5842; Fax: 412-624-4759; E-mail: spg1{at}pitt.edu.

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				T. C. Krzysiak, M. Grabe, and S. P. Gilbert Getting in Sync with Dimeric Eg5: INITIATION AND REGULATION OF THE PROCESSIVE RUN J. Biol. Chem., January 25, 2008; 283(4): 2078 - 2087. [Abstract] [Full Text] [PDF]