Release of isolated single kinesin molecules from microtubules

ABSTRACT: 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 microtubulebound 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 µM ADP displays an apparent second-order rate constant of 1.2 × 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 beadlabeled 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. Kinesin and its homologs are motor enzymes that use the free energy derived from ATP hydrolysis to drive the movement of membrane-bounded organelles, chromosomes, and other subcellular structures along cytoplasmic microtubules (1). Kinesin is a heterotetramer of two heavy chains and two light chains (2-4). The N-terminal ∼340 amino acids of each heavy chain form a compact globular "head" domain with dimensions 7 × 4.5 × 4.5 nm (5). Studies of truncated heavy chain derivatives demonstrated that the head contains a single ATPase catalytic site (6) and is sufficient to generate movement of the enzyme along microtubules A variety of experimental results demonstrate that kinesin and its two-headed derivatives are "processive" motors that maintain a continuous association with the microtubule for multiple catalytic turnovers and multiple mechanical steps: (i) the ability of kinesin molecules to follow the tracks of microtubule protofilaments during movement (13-16), (ii) the ability of single kinesin molecules to move continuously for hundreds of nanometers along the microtubule without dissociation (17-20), (iii) measurements demonstrating that the ATPase k cat /K 1/2,microtubule is larger than either the measured rate constant for the kinesin-microtubule association reaction or the calculated maximum diffusion-limited rate constant of that reaction (21-23), and (iv) demonstration that nucleotide-stimulated release of kinesin from the microtubule is slower than k cat and thus cannot be a step in the turnover cycle Movement of kinesin along the microtubule is thought to involve repeated microtubule binding and dissociation by individual kinesin heads, with the binding and dissociation reactions induced by changes in the chemical species bound at the catalytic sites. Heads bind tightly to the microtubule in the absence of nucleotide or in the presence of the substrate analog andenylyl imidodiphosphate (AMP-PNP