Kinetic studies of dimeric Ncd: evidence that Ncd is not processive.
Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. (1993) J. Biol. Chem. 268, 9005-9013]. Acid chemical quench flow methods were used to measure directly the rate of ATP hydrolysis, and stopped-flow kinetic methods were used to determine the kinetics of mantATP binding, mantADP release, dissociation of MC1 from the microtubule, and binding of MC1 to the microtubule. The results define a minimal kinetic mechanism, M.N + ATP M.N.ATP M.N.ADP.P N. ADP.P N.ADP + P M.N.ADP M.N + ADP, where N, M, and P represent Ncd, microtubules, and inorganic phosphate respectively, with k(+1) = 2.3 microM(-1) s(-1), k(+2) =23 s(-1), k(+3) =13 s(-1), k(+5)= 0.7 microM(-)(1) s(-)(1), and k(+6) = 3.7 s(-)(1). Phosphate release (k(+4)) was not measured directly although it is assumed to be fast relative to ADP release because Ncd is purified with ADP tightly bound at the active site. ATP hydrolysis occurs at 23 s(-)(1) prior to Ncd dissociation at 13 s(-)(1). The pathway for ATP-promoted detachment (steps 1-3) of Ncd from the microtubule is comparable to kinesin's. However, there are two major differences between the mechanisms of Ncd and kinesin. In contrast to kinesin, mantADP release for Ncd at 3.7 s(-)(1) is the slowest step in the pathway and is believed to limit steady-state turnover. Additionally, the burst amplitude observed in the pre-steady-state acid quench experiments is stoichiometric, indicating that Ncd, in contrast to kinesin, is not processive for ATP hydrolysis.
Foster
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Department of Biological Sciences, 518 Langley Hall, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Gilbert
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D019281:Dimerization; D029721:Drosophila Proteins; D004398:Dyneins / Q000378:metabolism; D006868:Hydrolysis; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D020409:Molecular Motor Proteins / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000378:metabolism
D029721:Drosophila Proteins; D020409:Molecular Motor Proteins; D011993:Recombinant Fusion Proteins; C066696:ncd protein, Drosophila; D062367:ortho-Aminobenzoates; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins; D004398:Dyneins; D016547:Kinesins