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,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-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 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,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.
Cochran
Jared C
JC
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Sontag
Christopher A
CA
Maliga
Zoltan
Z
Kapoor
Tarun M
TM
Correia
John J
JJ
Gilbert
Susan P
SP
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D002455:Cell Division; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D008956:Models, Chemical; D010725:Phosphocreatine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D014461:Ultracentrifugation
D029721:Drosophila Proteins; C063504:KAR3 protein, S cerevisiae; C470243:KIF11 protein, human; D008869:Microtubule-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; C066696:ncd protein, Drosophila; D010725:Phosphocreatine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins