Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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 mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2'-(3')-O-(N-methylanthraniloyl)-ATP and mant-ADP. The initial rapid equilibrium binding steps (1) and (6) are followed by isomerizations (k2 = 170 +/- 30 s-1 at 20 degrees C, k-5 greater than 100 s-1). The increase in fluorescence is 20-25% larger for K.T** than K.D*. The rate constant of the hydrolysis step k3 is 6-7 s-1. The fluorescence decreases after formation of K.T** at a rate of 7-10 s-1. This change could occur in step 3 or in step 4 if k4 much greater than k3. The value of k4 is larger than 0.1 s-1. The steady state rate is 0.003 s-1 which agrees with the rate of ADP dissociation (k5). Step 5 is rate limiting in the scheme in agreement with the conclusion of Hackney (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318) that ADP dissociation is the rate-limiting step.
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PMID:A kinetic study of the kinesin ATPase. 153 60

Addition of NaCl or KCl in the presence of 50 nM ATP induces a shift in the sedimentation coefficient (apparent S20,w) of kinesin from 9.4 S at low ionic strength to 6.5 S at high ionic strength. The midpoint for the transition occurs at ionic strength values of 0.39, 0.25, and 0.18 for pH values of 6.3, 6.9, and 8.3, respectively. Gel filtration experiments indicate that the transition to the 6.5 S species is accompanied by a decrease in the diffusion coefficient. Under all conditions which were tested, the 64-kDa beta subunits comigrate with the 120-kDa alpha subunits without any evidence for dissociation of the alpha 2 beta 2 complex. These results are consistent with the change in sedimentation coefficient being due to a conformational transition between a folded form at low ionic strength and an extended form at high ionic strength. This conformational transition is not significantly affected by the nature of the nucleotide bound at the active site since similar results are obtained both in the presence of excess EDTA, which removes the bound ADP, and after replacement of the bound ADP with adenosine 5'-(beta,gamma-imino)triphosphate. The alpha 2 form of kinesin, which lacks the beta subunits, undergoes a similar transition between a 6.7 S form at low ionic strength and a 5.1 S form at high ionic strength with a midpoint for the transition at an ionic strength of 0.5 at pH 6.9. Electron microscopic observation also indicates a transition between a folded conformation at low ionic strength and an extended conformation at high ionic strength for both the alpha 2 beta 2 and alpha 2 species.
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PMID:Kinesin undergoes a 9 S to 6 S conformational transition. 156 10

Bovine brain kinesin separates into two components on sucrose density gradient centrifugation. The predominant component is a heterotetramer of two 120 kDa alpha subunits and two 64 kDa beta subunits with an sedimentation coefficient of 9.6 S and a low Vm rate of microtubule-stimulated ATPase of 1.3 +/- 0.5 sec-1 at 25 degrees, pH 7.0. The minor element is a homodimer of two alpha subunits without beta subunits with a sedimentation coefficient of 6.9 S and a higher Vm rate of microtubule-stimulated ATPase of 7.0 +/- 1.9 sec-1. Microtubules stimulate the rate of release of ADP from the active site of the tetramer, but the rate of release is not fast enough to account for the rate of steady state ATP hydrolysis. Further complexity is indicated by biphasic release kinetics. In spite of the large difference in Vm ATPase rate for the two species, both drive the sliding of sea urchin axonemes over glass surfaces at the same velocity.
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PMID:Characterization of alpha 2 beta 2 and alpha 2 forms of kinesin. 182 68

Kinesin was isolated from bovine intradural nerve roots and conjugated with 5-(iodoacetamido)fluorescein. The modified kinesin (AF-kinesin) supports the movement of organelles along microtubules at rates comparable with those obtained using unmodified kinesin. AF-kinesin was purified by high-performance liquid chromatography. SDS/PAGE analysis of the purified fraction showed the presence of a fluorescent band at the position of the 125-kDa kinesin heavy chain. This protein promoted microtubule gliding with MgATP and with MgGTP at rates comparable to those of unlabelled kinesin. AF-kinesin had a fluorescein/protein ratio of one. Video microscopy at low light levels was used to monitor the interactions between the analogue and microtubules. AF-kinesin binds to microtubules in the presence of adenosine 5'-[beta, gamma-imino]triphosphate or ADP. Brief incubation of the microtubule. AF-kinesin complex with 10 mM ATP or GTP completely removes the labelled molecule. AF-kinesin can be inactivated in its ability to cause microtubule gliding by irradiating it with light that bleaches the bound fluorophore. When the protein is damaged in this way it still binds to microtubules and does so in the presence of ATP.
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PMID:Characterization of an active, fluorescein-labelled kinesin. 214 15

Determination of kinetic properties for kinesin adenosine triphosphatase (ATPase), a proposed motor for transport of membranous organelles, requires adequate amounts of kinesin with a consistent level of enzymatic activity. A purification procedure is detailed that produces approximately 2 mg of kinesin at up to 96% purity from 800 g of bovine brain. This protocol consists of a microtubule affinity step using 5'-adenylylimidodiphosphate (AMP-PNP); followed by gel filtration, ion exchange, and hydroxylapatite chromatography; and then sucrose density gradient centrifugation. The microtubule-activated ATPase activity of kinesin coeluted with kinesin polypeptides throughout the purification. Highly purified kinesin had a Vmax of 0.31 mumol/min/mg in the presence of microtubules, with a Km for ATP of 0.20 mM. The kinetic constants obtained in these studies compare favorably with physiological levels of ATP and microtubules. Variations in buffer conditions for the assay were found to affect ATPase activity significantly. A study of the ability of kinesin to utilize a variety of cation-ATP complexes indicated that kinesin is a microtubule-stimulated Mg-ATPase, but kinesin is able to hydrolyze Ca-ATP, Mn-ATP, and Co-ATP as well as Mg-ATP in the presence of microtubules. In the absence of microtubules, Ca-ATP appears to be the best substrate. Studies with several inhibitors of ATPases determined that vanadate inhibited kinesin ATPase at the lowest concentrations of inhibitor, but significant inhibition of the ATPase also occurred with submillimolar concentrations of AMP-PNP. Other inhibitors of kinesin include N-ethylmaleimide, adenosine diphosphate (ADP), pyrophosphate, and tripolyphosphate. Further characterization of the kinetic properties of the kinesin ATPase is important for understanding the molecular mechanisms for transport of membranous organelles along microtubules.
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PMID:Copurification of kinesin polypeptides with microtubule-stimulated Mg-ATPase activity and kinetic analysis of enzymatic properties. 252 82

Bovine brain kinesin binds ADP tightly and contains a stoichiometric amount of ADP at its active site when isolated in the presence of free Mg2+ (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318). EDTA in excess of Mg2+ weakens ADP binding and nucleotide-free kinesin can be prepared by gel filtration with excess EDTA. On addition of ATP, this nucleotide-free enzyme catalyzes the rapid hydrolysis of a stoichiometric amount of ATP in a burst phase followed by much slower continued ATP hydrolysis limited by the release of ADP from the active site. This burst reaction is evident both by formation of [32P]Pi from [gamma-32P]ATP and by formation of [alpha-32P]ADP from [alpha-32P]ATP. At 1.1 nM kinesin active sites, the observed rate of the burst phase increases linearly with ATP over the 1-20 nM range yielding a bimolecular rate of net ATP binding and hydrolysis of 2.5 microM-1 s-1. The intercept at zero ATP is 0.008 s-1 which equals the ADP release rate at 0.008-0.009 s-1. This predicts a Km for ATP of approximately 3.5 nM and measurements of the dependence on ATP concentration of the steady state rate and amount of bound ADP are consistent with a Km of this magnitude.
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PMID:Nucleotide-free kinesin hydrolyzes ATP with burst kinetics. 252 42

Kinesin from porcine brain was prepared by a procedure based on the strong binding of the protein to microtubules in the presence of sodium fluoride and ATP. The protocol reduces the requirement for taxol and AMP-PNP. The kinesin is active in terms of its ability to move microtubules on glass slides and its ATPase. The ATPase of this kinesin is about 8 nmol/min/mg; it is activated to 19 nmol/min/mg in the presence of microtubules. The relationship between gliding velocity and ATP concentration follows Michaelis-Menten kinetics. Using the motility assay, the maximal velocity is 0.78 micron/sec, and the Km value is 150 microM for ATP. For GTP the corresponding values are 0.38 micron/sec and 1.7 mM. ADP is a competitive inhibitor (Ki = 0.29 mM). Crude preparations of kinesin do not support motility on glass slides, whereas gel-filtered kinesin does. A search for potential inhibitory factors showed that one of them is MAP2; however, its inhibitory effect becomes visible only in certain conditions. MAP2 bound to microtubules does not inhibit kinesin-induced motility. However, when MAP2 and kinesin are preadsorbed to the glass surface independently of microtubules, MAP2 prevents the interaction of kinesin with microtubules, as if it formed a "lawn" that acted as a spacer and thus repelled the MAP-free microtubules or crosslinked the MAP-containing ones. The repelling effect of MAP2 domains (projection or assembly fragments obtained by chymotryptic cleavage) added separately is less pronounced and can be overcome by kinesin. These results reinforce the view of MAP2 as a spacer molecule.
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PMID:Interaction between kinesin, microtubules, and microtubule-associated protein 2. 253 84

Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms. Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm. The former "long pitch" helix is found only when RecA protein is bound to DNA. The latter "short pitch" helix is also observed in pure RecA protein polymers (also termed rods) and in the needle-like paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register. Addition of ATP or non-hydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers. These chains typically form closed, circular rings of 7(+/- 1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter. The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr "heat shock" ATPases. We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology.
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PMID:Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy. 269 35

The ATPase rate of kinesin isolated from bovine brain by the method of S.A. Kuznetsov and V.I. Gelfand [(1986) Proc. Natl. Acad. Sci. USA 83, 8530-8534)] is stimulated 1000-fold by interaction with tubulin (turnover rate per 120-kDa peptide increases from approximately equal to 0.009 sec-1 to 9 sec-1). The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that Pi release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (Ki less than 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by [14C]ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.
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PMID:Kinesin ATPase: rate-limiting ADP release. 297 Jun 38

The kinetic mechanism of the human kinesin ATPase motor domain K379, expressed in Escherichia coli, was determined by transient and steady-state kinetic studies. The steps in nucleotide binding were measured using the fluorescent substrate analogues, methylanthraniloyl ATP (mant-ATP) and mant-ADP. Both nucleotides gave a two-step fluorescence signal, an increase followed by a decrease, which indicates that two isomerizations are induced by nucleotide binding. The ATPase mechanism is fitted by a six-step reaction: [formula: see text] where, T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; K(T) and K(D) are states in rapid equilibrium with the free nucleotide. A set of kinetic constants for 20 degrees C 50 mM NaCl is K1 = 2 x 10(4) M-1, k2 = 200 s-1, k3 = 9 s-1, k5 = 0.01 s-1, and K6 = 2 x 10(-5) M. Values of K1 and K6 are estimates for mant-ATP and mant-ADP, respectively. ADP dissociation is the rate-limiting step. The rate constant for a decrease in fluorescence for the transitions from the high fluorescence K.T state to the low fluorescence K.D state is equal to k3, the rate constant of the hydrolysis step measured by quench flow experiments. The decrease could occur in step 3 or step 4 if k4 > k3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic mechanism of kinesin motor domain. 754 87


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