Kinetics and motility of the Eg5 microtubule motor.
We have investigated the kinetic properties of the slow plus end directed microtubule (MT) motor Eg5. The recombinantly expressed fusion protein E437GST, containing residues 12-437 of Eg5 fused to the N-terminus of glutathione S-transferase (GST), is dimeric and motile, translocating MTs at an average speed of 0.063 (+/-0.01) micrometers(-1). The kinetics of ATP turnover by E437GST were investigated using the fluorescent ATP analogue methylanthraniloyl-ATP (mantATP). In the absence of MTs, mantADP release from E437GST is slow (0.006 s(-1) in 50 mM NaCl) and rate-limiting. MTs accelerate this kinetic step approximately 850-fold to a maximal rate of 4.94 s(-1). Under these conditions, the steady-state rate of mantATP turnover was 1.92 s(-1), indicating that MT-activated mantADP release accounts for at least 40% of the total cycle time of the motor and is probably rate-limiting. This step is around 10-fold slower in Eg5 than in kinesin, consistent with it limiting the rate of physical stepping in both Eg5 and kinesin. The dissociation constants of the motor in the presence of various nucleotides were determined using MT pelleting assays. ADP stabilizes the weakest bound state of the motor, while ATP, ATP gamma S, AMPPNP, and apyrase all induce a shift toward tighter binding states. Overall, the data indicate that Eg5 displays strong kinetic homologies with the two other well-characterized MT motors, kinesin and non claret disjunctional, suggesting that all kinesin superfamily motors may share the same basic mechanochemistry.
Lockhart
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Molecular Motors Group, Marie Curie Research Institute, Oxted, Surrey, UK.
Cross
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D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D001483:Base Sequence; D017931:DNA Primers; D005456:Fluorescent Dyes; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008870:Microtubules / Q000737:chemistry*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D029867:Xenopus Proteins; D062367:ortho-Aminobenzoates / Q000737:chemistry
D017931:DNA Primers; D005456:Fluorescent Dyes; C088335:KIF11 protein, Xenopus; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D062367:ortho-Aminobenzoates; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D016547:Kinesins