Mechanistic analysis of the mitotic kinesin Eg5

Abstract

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: kcat = 5.5 s -1, K1/2,Mt = 0.7 μM, and Km,ATP = 25 μM; and for Eg5-437: kcat = 2.9 s-1, K1/2Mt = 4.5 μM, and Km,ATP = 19 μM. 2′(3′)-O-(N- Methylarathraniloyl)-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 K1/2,Mt and the mantADP release K1/2,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.