Mechanistic analysis of the mitotic kinesin Eg5

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,K-Mt=0.7 muM, and K-m,K-ATP=25 muM; and for Eg5-437: k(cat)=2.9 s(-1), K-1/2,K-Mt=4.5 muM, and K-m,K-ATP=19 muM. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 muM(-1) s(-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 muM(-1) s(-1)) than Eg5-437 (0.7 muM(-1) s(-1)), consistent with the steady-state K-1/2,K-Mt and the mantADP release K-1/2,K-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.