Kinetic mechanism of a monomeric kinesin construct.
The kinetic mechanism is analyzed for a monomeric human kinesin construct K332. In the absence of microtubules, the rate constants of the ATPase cycle are very similar to dimeric human kinesin K379 and whole kinesin from bovine brain. The microtubule-activated ATPase is 60 s(-1) at 20 degrees C; Km(Mt) is 5 microM; dissociation constants in the presence of ATP and ADP are 9 microM and 16 microM, respectively. The values of dissociation constants are 5 times larger than for K379. Binding of K332 to microtubules increased the rate of the hydrolysis step from 7 s(-1) to greater than 200 s(-1) and the 2'-(3')-O-(N-methylanthraniloyl) (mant) ADP dissociation step from 0.02 s(-1) to greater than 100 s(-1). At higher ionic strength, more than one ATP is hydrolyzed before dissociation of MtK332 (small processivity). Data are fitted to the kinetic scheme. [equation: see text] Approximate values of rate constants are k1 = 500 s(-1), k2 > or = 200 s(-1), k3k4/(k3 + k4) = 100 s(-1), k(dis) = 80+/-10 s(-1). Two experiments to measure k4 gave 110 s(-1) from the maximum rate of dissociation of mant ADP for reaction of K x ADP with microtubules and 300 s(-1) from extrapolation to zero concentration of rate of binding of mant ADP to MtK. It is proposed that mant ADP dissociation is a two-step process. In the simple scheme, k4 is the effective rate of the two-step release of ADP, k4 = 150 s(-1) to 200 s(-1), and k3 = 150 s(-1) to 200 s(-1) to account for the steady state rate.
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Department of Molecular Genetics and Cell Biology, The University of Chicago, Illinois 60637, USA.
Taylor
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D006801:Humans; D016547:Kinesins / Q000737:chemistry*; D007700:Kinetics; D008870:Microtubules / Q000737:chemistry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins