Nucleotide-free Kinesin Hydrolyzes ATP with Burst Kinetics

Hackney, David D.; Malik, Abds-Sami; Wright, Kwame-Lante

doi:10.1016/s0021-9258(18)71570-6

Cited by 37 publications

(23 citation statements)

References 15 publications

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“…The net binding of ADP to kinesin is considerably weaker in the presence of excess EDTA (26), but net binding still occurs if the free mant-ADP level is sufficiently high. In the experiment of Figure 7, the K349‚Mgmant-ADP complex was equilibrated with free mant-ADP for varying times in the presence of excess EDTA and then chased with a large excess of ADP.…”

Section: Characterization Of Mant-adp Release Usingmentioning

confidence: 98%

“…Detailed analysis of the nucleotide binding of DKH357 is complicated by the instability of nucleotide-free Drosophila kinesin, which undergoes a transition to a conformation that is not capable of rapidly rebinding ATP (24). Nucleotide-free kinesin from bovine (26) and human (20) sources, however, retain the ability to rapidly rebind ATP, and some comparative studies with human K349 were also performed. Initial experiments with human K349 indicated that its kinetics were highly similar to those of Drosophila DKH357, which terminates at the homologous position.…”

Section: Characterization Of Mant-adp Release Usingmentioning

confidence: 99%

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Interaction of Mant-Adenosine Nucleotides and Magnesium with Kinesin

1998

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Displacement of the fluorescent substrate analogue methylanthraniloyl ADP (mant-ADP) from kinesin by excess ATP results in a biphasic fluorescent transient. The pH and microtubule dependence of the rates and amplitudes indicates that the two phases are produced by release of bound mant-ADP, with an excess of the 3'-isomer, followed by the subsequent relaxation of the free 2'- and 3'-isomers to their equilibrium distribution. The first phase for release of mant-ADP is accelerated by microtubules and occurs at the same rate as ADP release measured using [32P]ADP. The second phase is subject to base catalysis and occurs at the same rate as the isomerization of isolated 2'- or 3'-mant-ATP over a 100-fold range of rates. The bound mant-ADP isomers undergo isomerization rapidly when bound to kinesin at pH 8.2, whereas mant-ADP isomers interconvert only slowly when bound to myosin. No fluorescence resonance energy transfer occurs between the single tryptophan in the kinesin neck domain and bound mant-ADP, but efficient energy transfer does occur from protein tyrosine groups. The rate of mant-ADP release in the absence of microtubules is minimal (0.005 s-1) at pH 7-8, 2 mM Mg2+, and 25 mM KCl but is accelerated at lower pH (0.04 s-1 at pH 5.5) and either lower or higher [KCl] (0.01 and 0. 06 s-1 at 0 and 800 mM KCl, respectively). The microtubule-stimulated rate of ADP release is accelerated at low pH and inhibited by high concentrations of monovalent salts. Reduction of the free Mg2+ by addition of excess EDTA increases the release of mant-ADP from E.MgADP to 0.03 s-1. This acceleration at low Mg2+ likely represents sequential release of Mg2+ at 0.03 s-1 followed by rapid release of ADP, as the rate of ADP release from Mg-free E.ADP is fast (>0.5 s-1). At high Mg2+, rebinding of Mg2+ to E.ADP forces release of ADP from the E.MgADP complex at 0.005 s-1.

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Section: Characterization Of Mant-adp Release Usingmentioning

confidence: 98%

Section: Characterization Of Mant-adp Release Usingmentioning

confidence: 99%

Interaction of Mant-Adenosine Nucleotides and Magnesium with Kinesin

1998

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“…Dissociation Constant of K413 and K332⅐ADP for Microtubules-Previous kinetic studies on intact brain kinesin and on truncated constructs (Hackney, 1988;Hackney et al, 1989;Sadhu and Taylor, 1992;Gilbert and Johnson, 1994) indicated that the rate-limiting step in the microtubule kinesin ATPase cycle was ADP release, implying that kinesin⅐ADP would be the predominant species under steady-state conditions. To test this with the human construct, the affinities of tritium-labeled FIG.…”

Section: Characterization Of Human Kinesin Constructs-previousmentioning

confidence: 99%

Equilibrium Studies of Kinesin-Nucleotide Intermediates

Rosenfeld

Rener

Correia

et al. 1996

Journal of Biological Chemistry

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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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“…As we study these newly discovered motors in detail, the results should provide information to understand the common mechanistic features that are required to couple nucleotide hydrolysis to force production to begin to define the variations that result in the functional and motility differences observed. Kinesin is the first of the cytoplasmic motors to be investigated using pre-steady-state kinetic techniques (Hackney, 1988;Hackney et al, 1989;Sadhu & Taylor, 1992). The initial kinesin mechanistic experiments have focused on the native bovine brain kinesin preparation studied in the absence of microtubules.…”

mentioning

confidence: 99%

“…The initial kinesin mechanistic experiments have focused on the native bovine brain kinesin preparation studied in the absence of microtubules. Hackney et al (1988Hackney et al ( ,1989 demonstrated that ADP is tightly bound such that kinesin when purified contains a stoichiometric amount of ADP bound at the active site. Furthermore, it is ADP release rather than phosphate release that is rate limiting during steady-state turnover, and the rate of ADP release is increased by the addition of microtubules.…”

mentioning

confidence: 99%

Pre-Steady-State Kinetics of the Microtubule.cntdot.Kinesin ATPase

Gilbert¹,

Johnson²

1994

Biochemistry

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The pre-steady-state kinetics of the microtubule-kinesin ATPase were investigated by chemical-quench flow methods using the Drosophila kinesin motor domain (K401) expressed in Escherichia coli [Gilbert, S. P., & Johnson, K. A. (1993) Biochemistry 32, 4677-4684]. The results define a minimal mechanism: M.K + ATP in equilibrium with (M).K.ATP in equilibrium with (M).K.ADP.Pi in equilibrium with M.K.ADP + Pi in equilibrium with M.K + ADP, where M, K, and Pi represent microtubules, kinesin, and inorganic phosphate, respectively, with k+1 = 0.8-3 microM-1 s-1, k-1 = 100-300 s-1, k+2 = 70-120 s-1, k+4 = 10-20 s-1, and k+3 >> k-2 and k+3 >> k+4. Conditions were as follows: 25 degrees C, 20 mM HEPES, pH 7.2 with KOH, 5 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM potassium acetate, 1 mM DTT. The experiments presented do not determine the step in the cycle where kinesin dissociates from the microtubule or the step at which kinesin reassociates with the microtubule; therefore, the steps that may represent kinesin as the free enzyme are indicated by (M). A burst of ADP product formation was observed during the first turnover of the enzyme in the acid-quench experiments that define the ATP hydrolysis transient. The observation of the burst demonstrates that product release is rate limiting even in the presence of saturating microtubule concentrations. The pulse-chase experiments define the time course of ATP binding to the microtubule-K401 complex. At low ATP concentrations, ATP binding limits the rate of the burst. However, at high concentrations of ATP, ATP binding is faster than the rate of ATP hydrolysis with k+2 = 70-120 s-1. The amplitude of the burst of the ATP binding transient reached a maximum of 0.7 per site at saturating concentrations of ATP and microtubules. The amplitude of less than 1 is attributed to the fast k(off) for ATP (k-1 = 100-300 s-1) that leads to a partitioning of the M.K.ATP complex between ATP hydrolysis (k+2) and ATP release (k-1). These results indicate that ATP binds weakly to the M.K complex (Kd,ATP app approximately 100 microM). ADP release (k+4 = 10-20 s-1) is rate limiting during steady-state turnover, indicating that microtubules activate the kinesin ATPase by increasing k(off),ADP from 0.01 s-1 in the absence of microtubules to 10-20 s-1 at saturating microtubule concentrations.

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Nucleotide-free Kinesin Hydrolyzes ATP with Burst Kinetics

Cited by 37 publications

References 15 publications

Interaction of Mant-Adenosine Nucleotides and Magnesium with Kinesin

Interaction of Mant-Adenosine Nucleotides and Magnesium with Kinesin

Equilibrium Studies of Kinesin-Nucleotide Intermediates

Pre-Steady-State Kinetics of the Microtubule.cntdot.Kinesin ATPase

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