Release of isolated single kinesin molecules from microtubules.
Previous studies on the motor enzyme kinesin suggesting that the enzyme molecule tightly binds to a microtubule by only one of its two mechanochemical head domains were performed with multiple kinesin molecules on each microtubule, raising the possibility that interactions between adjacent bound molecules may interfere with the binding of the second head. To characterize the microtubule-bound state of isolated single kinesin molecules, we have measured the rates of nucleotide-induced dissociation of the complex between microtubules and bead-labeled single molecules of the dimeric kinesin derivative K448-BIO using novel single-molecule kinetic methods. Complex dissociation by <2 microM ADP displays an apparent second-order rate constant of 1.2 x 10(4) M-1 s-1. The data suggest that only one of the two heads is bound to the microtubule in the absence of ATP, that binding of a single ADP to the complex is sufficient to induce dissociation, and that even lengthy exposure of kinesin to the microtubule fails to produce significant amounts of a two-head-bound state under the conditions used. The inhibitor adenylyl imidodiphosphate (AMP-PNP) induces stochastic pauses in the movement of bead-labeled enzyme molecules in 1 mM ATP. Exit from pauses occurs at 2 s-1 independent of AMP-PNP concentration. The same rate constant is obtained for dissociation of the transient kinesin-microtubule complexes formed in 1 mM ADP, 0.5 mM AMP-PNP, suggesting that release of a single AMP-PNP molecule from the enzyme is the common rate-limiting step of the two processes. The results are consistent with alternating-sites movement mechanisms in which two-head-bound states do not occur in the enzyme catalytic cycle until after ATP binding.
Vugmeyster
Y
Y
Department of Biochemistry and The Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02254, USA.
Berliner
E
E
Gelles
J
J
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000465:Algorithms; D000818:Animals; D002417:Cattle; D004330:Drosophila; D016547:Kinesins / Q000378:metabolism* / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D009068:Movement; D011485:Protein Binding / Q000187:drug effects
D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins