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Query: EC:3.6.4.4 (kinesin)
5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The head or motor domain of the ncd (non-claret disjunctional) molecular motor is 41% identical to that of kinesin, yet moves along microtubules in the opposite direction to kinesin. We show here that despite the reversed directionality of ncd, its kinetics in solution are homologous in key respects to those of kinesin. The rate limiting step, ADP release, occurs at 0.0033 s-1 at 100 mM NaCl and is accelerated approximately 1000-fold when the motor binds to microtubules. Other reaction steps are all very fast (> 0.1 s-1) compared with ADP release, and the motor is consequently paused in the ncd.ADP state until microtubule binding occurs (Kd = 2 microM), at which point ADP release is triggered and the motor locks onto the microtubule in a rigor-like state. These data identify close functional homology between the strong binding states of kinesin and ncd, and in view of this we discuss a possible mechanism for directional reversal, in which the strong binding states of ncd and kinesin are functionally identical, but the weak binding states are biased in opposite directions.
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PMID:Origins of reversed directionality in the ncd molecular motor. 811 90

A truncated motor domain of the alpha subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH340) extends from the N terminus to amino acid 340. The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis of 0.029 +/- 0.002 s-1 in the absence of microtubules. The rate of release of bound ADP is 0.026 +/- 0.003 s-1. The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules. The rate of ATP hydrolysis is stimulated 3000 fold-by addition of microtubules (MT) (kcat = 80 s-1; KMT0.5,ATPase = 160 nM for half-saturation of the ATPase rate by microtubules at saturating ATP levels; KMT0.5ATPase = 43 microns for half-saturation of the ATPase rate by ATP at saturating microtubule levels). Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)t-riphosphate. One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules. In the presence of MgATP, KMT0.5,Binding for physical binding of DKH340 to microtubules is weaker than KMT0.5,ATPase for stimulation of hydrolysis. These results are consistent with a model in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule. For this model, the kcat/KMT0.5,ATPase ratio of 5 x 10(8) M-1 s-1 is at least as large as the bimolecular rate constant for association with microtubules, and this value approaches the diffusion controlled limit. Nucleotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgATP (t1/2 > or = 10 min), and thus it is likely to be in a conformational state which is not produced during steady state ATP hydrolysis.
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PMID:Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization. 820 59

DKH392 is a construct which contains the first 392 amino acids of the alpha-subunit of Drosophila kinesin and is dimeric in solution (Huang, T.-G., Suhan, J., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16502-16507). The ATPase rate of DKH392 was 0.005 s-1 in the absence of MTs. One ADP bound tightly to each subunit and the release of this ADP was the rate-limiting step in ATP hydrolysis. Microtubules accelerated the rate of ADP release and increased the rate of steady state ATP hydrolysis by almost 10,000-fold (kcat = approximately 45 s-1). The KMT0.5,ATPase value for saturation of the stimulation of the ATPase reaction by microtubules was 50 nM at 8 nM DKH392, but decreased at lower concentrations of DKH392. Physical binding of DKH392 to microtubules in the presence of 1 mM MgATP paralleled saturation of the stimulation of the ATPase activity by microtubules indicating that the rate-limiting step in microtubule-stimulated ATP hydrolysis occurs while DKH392 is bound to the microtubule. These results suggest that microtubule-stimulated ATP hydrolysis by DKH392 may be processive with the hydrolysis of multiple ATP molecules during each diffusional encounter of DKH392 with a microtubule.
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PMID:The rate-limiting step in microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule. 820 61

Motor proteins move unidirectionally along cytoskeletal polymers by coupling translocation to cycles of ATP hydrolysis. The energy from ATP is required both to generate force and to dissociate the motor-filament complex in order to begin a new chemomechanical cycle. For myosin, force production is associated with phosphate release following ATP hydrolysis, whereas dissociation of actomyosin is tightly coupled to the binding of ATP. Dynein, a microtubule motor, uses a similar cycle, suggesting that all cytoskeletal motors might operate by a common mechanism. Here we investigate kinesin's chemomechanical cycle by assaying microtubule movement by single kinesin molecules when intermediate states in the hydrolysis cycle are prolonged with ATP analogues or inhibitors. In contrast to myosin and dynein, kinesin with bound ADP dissociates from microtubules during translocation, whereas kinesin with unhydrolysed nucleotide remains tightly associated with the polymer. These findings imply that kinesin converts ATP energy into mechanical work by a pathway distinct from that of myosin or dynein.
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PMID:Chemomechanical cycle of kinesin differs from that of myosin. 842 15

The Drosophila kinesin heavy-chain gene was truncated to obtain the N-terminal 401 amino acid motor domain (designated K401) containing both the microtubule and ATP binding sites. The plasmid construct with the truncated kinesin gene was used to transform Escherichia coli. After induction, K401 was expressed as soluble kinesin protein at high levels and purified to homogeneity in milligram quantities. The purified protein was active and behaved as native kinesin with respect to its steady-state kinetic properties: K401 demonstrated a very low ATPase activity (kcat = 0.01 s-1) which was stimulated approximately 1000-fold by the addition of microtubules (kcat = 10 s-1; K0.5,MT = 0.9 microM tubulin; Km,ATP = 31 microM). Like native kinesin, K401 when purified contained ADP tightly bound at its active site, and the release of ADP from the active site occurred at a rate equal to the steady-state ATPase kcat. Active-site measurements using [alpha-32P]ATP demonstrated a stoichiometry of one ATPase site per K401 molecule. Like native kinesin, K401 can also hydrolyze MgGTP, and in the presence of microtubules, the rate of hydrolysis was increased dramatically from 0.03 to 16 s-1 (K0.5,MT = 2 microM tubulin; Km,GTP = 3.5 mM). These results establish that an active kinesin motor domain can be bacterially expressed and that this domain, the N-terminal 401 amino acids of the Drosophila kinesin heavy chain without light chains or additional eukaryotic factors, has full catalytic activity with microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression, purification, and characterization of the Drosophila kinesin motor domain produced in Escherichia coli. 848 45

Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy-chain phosphorylation, and (d) ADP release is fast. Thus, during steady-state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily, other than myosin-II, providing the opportunity to obtain insights about the evolution of all myosin isoforms.
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PMID:Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase. 860 84

Kinesin is the founding member of a superfamily of microtubule based motor proteins that perform force-generating tasks such as organelle transport and chromosome segregation. It has two identical approximately 960-amino-acid chains containing an amino-terminal globular motor domain, a central alpha-helical region that enables dimer formation through a coiled-coil, and a carboxy-terminal tail domain that binds light chains and possibly an organelle receptor. The kinesin motor domain of approximately 340 amino acids, which can produce movement in vitro, is much smaller than that of myosin (approximately 850 amino acids) and dynein (1,000 amino acids), and is the smallest known molecular motor. Here, we report the crystal structure of the human kinesin motor domain with bound ADP determined to 1.8-A resolution by X-ray crystallography. The motor consists primarily of a single alpha/beta arrowhead-shaped domain with dimensions of 70 x 45 x 45 A. Unexpectedly, it has a striking structural similarity to the core of the catalytic domain of the actin-based motor myosin. Although kinesin and myosin have virtually no amino-acid sequence++ identity, and exhibit distinct enzymatic and motile properties, our results suggest that these two classes of mechanochemical enzymes evolved from a common ancestor and share a similar force-generating strategy.
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PMID:Crystal structure of the kinesin motor domain reveals a structural similarity to myosin. 860 61

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.
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PMID:Equilibrium studies of kinesin-nucleotide intermediates. 862 18

Neurospora kinesin (Nkin) is a distant relative of the family of conventional kinesins, members of which have been identified in various animal species. As in its animal counterparts, Nkin most likely is an organelle motor. Because it is a functional homologue of the kinesin heavy chain of higher eukaryotes, its biophysical and motility properties were compared with those of other conventional kinesins. Purified Nkin behaves as a homodimeric complex composed of two subunits of a 105-kDa polypeptide. Based on its hydrodynamic properties (Stokes radius and sedimentation coefficient), Nkin is an elongated molecule, although it is more compact than its animal counterparts. A detailed comparison of the motility properties of Nkin with those of animal conventional kinesins reveals similarities and some intriguing differences. Nkin is less effective than other kinesins in the use of natural nucleoside triphosphates but responds to a selection of ATP analogues in a similar fashion as mammalian kinesin. Even in the presence of saturating concentrations of ATP, Nkin is significantly more sensitive to ADP or tripolyphosphate than other kinesins. Both the ATP-driven microtubule gliding activity and the microtubule-stimulated ATPase activity of Nkin obey Michaelis-Menten kinetics. Surprisingly, however, the Km values for both these activities are approximately an order of magnitude higher than those of other kinesins. Whether the low affinity for ATP suggested by these high Km values is related to the high rate of motility remains to be determined.
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PMID:Characterization of the biophysical and motility properties of kinesin from the fungus Neurospora crassa. 863 82

Kinesin superfamily molecular motors step along microtubules (MTs) via a cycle of conformational changes which is coupled to ATP turnover. To probe the coupling mechanism, we titrated the effects of various nucleotides on MT binding by two superfamily members; MT plus-end-directed kinesin and MT minus-end-directed non claret disjunctional (ncd). For both motors, the nucleotide-free state induced by apyrase was the strongest binding (K(kin)d approximately 0.003 micro M, K(ncd)d approximately 0.24 micro M), whilst the ADp state was the weakest binding (K(kin)d approximately 11.32 micro M, K(ncd)d approximately 12.02 micro M). In ATP, the motor. ADP state dominates and the binding is accordingly ADP-like, but in the presence of the slowly hydrolysed analogue adenosine 5'-O-(3-thiotriphosphate) there is a shift towards tighter binding (K(kin)d approximately 4.23 micro M, K(ncd)d approximately 2.34 micro M), consistent with a tight-binding motor. ATP-like state being enriched. In the presence of non-hydrolysable analogue beta,gamma-imidoadenosine 5'-triphosphate the binding is still tighter (K(kin)d approximately <0.27 micro M, K(ncd)d approximately 0.21 micro M), close to the values obtained with apyrase. For both kinesin and ncd, ADP has the unique quality that it traps the motor in a weak binding state. MT tight binding catalyses escape from this state, changing the active site conformation such that both ADP release and ADP binding are accelerated. The data are consistent with a simple two-state scheme in which both kinesis and ncd switch from weak to strong binding via ADP release, and back again via ADP trapping. In a two-state model, the transition from weak to strong binding is force-generating.
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PMID:Weak and strong states of kinesin and ncd. 863 60


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