ATPase mechanism of Eg5 in the absence of microtubules: insight into microtubule activation and allosteric inhibition by monastrol.
The ATPase mechanism of kinesin superfamily members in the absence of microtubules remains largely 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. We have used steady-state and presteady-state kinetics to define the minimal ATPase mechanism for apoEg5 in the absence and presence of the Eg5-specific inhibitor, monastrol. ATP and ADP binding both occur via 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 of these steps is limited by the relatively slow isomerization of the Eg5-ATP collision complex. A conformational change coupled to ADP release is the rate-limiting step in the pathway. We propose that the microtubule amplifies and accelerates the structural transitions needed to form the ATP hydrolysis competent state and for rapid ADP release, thus stimulating ATP turnover and increasing enzymatic efficiency. Monastrol appears to bind weakly to the Eg5-ATP collision complex, but after tight ATP binding, the affinity for monastrol increases, thus inhibiting the conformational change required for ADP product 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 the active site during the ATPase cycle.
Cochran
Jared C
JC
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Gilbert
Susan P
SP
D020106:Acrylamide / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000494:Allosteric Regulation; D004789:Enzyme Activation; D006868:Hydrolysis; D016547:Kinesins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism*; D011487:Protein Conformation; D011743:Pyrimidines / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013871:Thiones / Q000494:pharmacology*
C470243:KIF11 protein, human; D011743:Pyrimidines; D013871:Thiones; D020106:Acrylamide; C400223:monastrol; D000251:Adenosine Triphosphatases; D016547:Kinesins