Equilibrium studies of kinesin-nucleotide intermediates.
We have examined the energetics of the interactions of two kinesin constructs with nucleotide and microtubules to develop a structural model of kinesin-dependent motility. Dimerization of the constructs was found to reduce the maximum rate of the microtubule-activated kinesin ATPase 5-fold. Beryllium fluoride and aluminum fluoride also reduce this rate, and they increase the affinity of kinesin for microtubules. By contrast, inorganic phosphate reduces the affinity of a dimeric kinesin construct for microtubules. These findings are consistent with a model in which the kinesin head can assume one of two conformations, "strong" or "weak" binding, determined by the nature of the nucleotide that occupies the active site. Data for dimeric kinesin are consistent with a model in which kinesin.ATP binds to the microtubule in a strong state with positive cooperativity; hydrolysis of ATP to ADP+P(i) leads to dissociation of one of the attached heads and converts the second, attached head to a weak state; and dissociation of phosphate allows the second head to reattach. These results also argue that a large free energy change is associated with formation of kinesin.ADP.P(i) and that this step is the major pathway for dissociation of kinesin from the microtubule.
Rosenfeld
S S
SS
Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Rener
B
B
Correia
J J
JJ
Mayo
M S
MS
Cheung
H C
HC
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008870:Microtubules / Q000502:physiology; D008961:Models, Structural; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D017931:DNA Primers; D010710:Phosphates; D011994:Recombinant Proteins; D014404:Tubulin; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins