| Abstract
| - The ATPase mechanism of kinesin superfamily members in the absence of microtubules remainslargely uncharacterized. We have adopted a strategy to purify monomeric human Eg5 (HsKSP/Kinesin-5) in the nucleotide-free state (apoEg5) in order to perform a detailed transient state kinetic analysis. Wehave used steady-state and presteady-state kinetics to define the minimal ATPase mechanism for apoEg5in the absence and presence of the Eg5-specific inhibitor, monastrol. ATP and ADP binding both occurvia a two-step process with the isomerization of the collision complex limiting each forward reaction.ATP hydrolysis and phosphate product release are rapid steps in the mechanism, and the observed rate ofthese steps is limited by the relatively slow isomerization of the Eg5−ATP collision complex. Aconformational change coupled to ADP release is the rate-limiting step in the pathway. We propose thatthe microtubule amplifies and accelerates the structural transitions needed to form the ATP hydrolysiscompetent state and for rapid ADP release, thus stimulating ATP turnover and increasing enzymaticefficiency. Monastrol appears to bind weakly to the Eg5−ATP collision complex, but after tight ATPbinding, the affinity for monastrol increases, thus inhibiting the conformational change required for ADPproduct release. Taken together, we hypothesize that loop L5 of Eg5 undergoes an “open” to “closed”structural transition that correlates with the rearrangements of the switch-1 and switch-2 regions at theactive site during the ATPase cycle.
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