Equilibrium Studies of Kinesin-Nucleotide Intermediates

Rosenfeld, Steven S.; Rener, Brenda; Correia, John J.; Mayo, Matthew S.; Cheung, Herbert C.

doi:10.1074/jbc.271.16.9473

Cited by 49 publications

(73 citation statements)

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“…However, they used a concentration of AMPPNP in their experiments 8-fold lower than in ours. We note that AMPPNP has a relatively weak affinity for kinesin (31). This raises the possibility that in the presence of MTs, a substantial proportion of MD might have been in a rigor state.…”

Section: Small Molecule Inhibitors Provide Insight Into the Physiologmentioning

confidence: 83%

“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

Kaan

Major

Tkocz

et al. 2013

Journal of Biological Chemistry

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Section: Small Molecule Inhibitors Provide Insight Into the Physiologmentioning

confidence: 83%

“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

Kaan

Major

Tkocz

et al. 2013

Journal of Biological Chemistry

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“…Structural and kinetic studies of myosin have shown that ADP⅐BeF x acts as an ATP analog, much as does adenylyl-imidodiphosphate (AMP-PNP) (28)(29)(30). Kinesin associates tightly with MTs when bound to ADP⅐BeF x (31)(32)(33), exactly as for AMP-PNP or ATP (34)(35)(36)(37)(38)(39). Moreover, complexes of kinesin with MTs generate identical EPR spectra in the presence of AMP-PNP or ADP⅐BeF x (40).…”

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confidence: 99%

Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain

Guydosh

Block

2006

Proc. Natl. Acad. Sci. U.S.A.

128

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The two-headed kinesin motor harnesses the energy of ATP hydrolysis to take 8-nm steps, walking processively along a microtubule, alternately stepping with each of its catalytic heads in a hand-over-hand fashion. Two persistent challenges for models of kinesin motility are to explain how the two heads are coordinated (''gated'') and when the translocation step occurs relative to other events in the mechanochemical reaction cycle. To investigate these questions, we used a precision optical trap to measure the singlemolecule kinetics of kinesin in the presence of substrate analogs beryllium fluoride or adenylyl-imidodiphosphate. We found that normal stepping patterns were interspersed with long pauses induced by analog binding, and that these pauses were interrupted by short-lived backsteps. After a pause, processive stepping could only resume once the kinesin molecule took an obligatory, terminal backstep, exchanging the positions of its front and rear heads, presumably to allow release of the bound analog from the new front head. Preferential release from the front head implies that the kinetics of the two heads are differentially affected when both are bound to the microtubule, presumably by internal strain that is responsible for the gating. Furthermore, we found that ATP binding was required to reinitiate processive stepping after the terminal backstep. Together, our results support stepping models in which ATP binding triggers the mechanical step and the front head is gated by strain.motor coordination ͉ optical tweezers ͉ single-molecule biophysics ͉ gating ͉ processivity C onventional kinesin, the founding member of the Kinesin-1 family, uses energy from ATP hydrolysis to transport cellular cargo, taking 8-nm steps along microtubules (MTs) (1). Kinesin molecules are formed from two identical heavy chains, whose N-terminal regions fold to form catalytic motor domains, or heads, which are joined by ''neck-linker'' regions to a common, coiled-coil stalk. With each step, the two kinesin heads exchange leading and trailing positions as they alternately hydrolyze ATP, generating ''hand-over-hand'' motion (2-7). This motion is processive under moderate loads for up to Ϸ100 steps in succession, a property that enables small numbers of motors to ferry cargo over long distances (8, 9). The mechanism responsible for the remarkable processivity of kinesin remains unresolved. Furthermore, the relative order of the translocation step with respect to other steps in the mechanochemical cycle is not established.A corollary of kinesin processivity is that its two heads do not function independently, but in concert. Put another way, the relative phase between the heads must be synchronized by a gating mechanism that prevents both heads from dissociating simultaneously from the MT. Because of the 2-fold symmetry imparted by the kinesin structure, this mechanism must break symmetry to permit synchronization. The most attractive candidate for such a mechanism is the mechanical strain that develops when the two heads separate an...

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“…Hydrolysis of ATP by kinesin proceeds through attack of water on the ␥ phosphoryl to yield P i that contains one water-derived oxygen and three oxygens from the nonbridge ␥ oxygens of the ATP, as indicated in Scheme 2 for hydrolysis of unenriched ATP in 100% 18 O-enriched water. If P i release via k 3 is much faster than resynthesis of ATP via k Ϫ2 , then the released P i will have one and only one water-derived 18 O oxygen.…”

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“…If P i release via k 3 is much faster than resynthesis of ATP via k Ϫ2 , then the released P i will have one and only one water-derived 18 O oxygen. If reversal to reform ATP does occur, then the ATP that is formed will retain the water-derived 18 O oxygen 75% of the time for a random process.…”

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The tethered motor domain of a kinesin-microtubule complex catalyzes reversible synthesis of bound ATP

Hackney

2005

Proc. Natl. Acad. Sci. U.S.A.

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Although the steps for the forward reaction of ATP hydrolysis by the motor protein kinesin have been studied extensively, the rates for the reverse reactions and thus the energy changes at each step are not as well defined. Oxygen isotopic exchange between water and P i was used to evaluate the reverse rates. The fraction of the kinesin⅐ADP⅐P i complex that reverts to ATP before release of Pi during net hydrolysis was Ϸ0 and Ϸ2.6% in the absence and presence of microtubules (MTs), respectively. The rate of synthesis of bound ATP from free P i and the MT⅐kinesin⅐ADP complex was Ϸ1.7 M ؊1 ⅐s ؊1 (K0.5 ADP ‫؍‬ 70 M) with monomeric kinesin in the absence of net hydrolysis. Synthesis of bound ATP from the ADP of the tethered head of a dimer-MT complex was 20-fold faster than for the monomer-MT complex. This MT-activated ATP synthesis at the tethered head is in marked contrast to the lack of MT stimulation of ADP release from the same site. The more rapid ATP synthesis with dimers suggests that the tethered head binds behind the strongly attached head, because this positions the neck linker of the tethered head toward the plus end of the MT and would thus facilitate its docking on synthesis of ATP. The observed rate of ATP synthesis also puts limits on the overall energetics that suggest that a significant fraction of the free energy of ATP hydrolysis is available to drive the docking of the neck linker on binding of ATP.ATPase ͉ energy coupling ͉ motility ͉ motor protein ͉ isotopic exchange K inesin-1 is molecular motor that moves along microtubules (MTs) in a highly processive manner. Scheme 1 presents a minimal mechanism for ATP hydrolysis by the MT complex of a kinesin monomer motor domain (see refs. 1-3 for recent reviews of the ATPase mechanism and coupling to motility). ATP binds rapidly and is hydrolyzed at 100-300 s Ϫ1 . P i release occurs without a lag after hydrolysis, and thus P i release is at least as fast as hydrolysis (k 3 Ն k 2 ). ADP release is partially ratelimiting for conventional kinesin-1 and may be the principal rate limiting step for some other kinesin superfamily members.Another important aspect, however, is the equilibrium constant of each step, because it determines the free energy that is available at that step for coupling to movement against a load. The equilibrium constants can be determined if the rates in the reverse direction are known, but this is difficult because the overall equilibrium strongly favors hydrolysis. Oxygen isotopic exchange is a powerful technique for evaluation of the reversibility of the hydrolysis reaction and has provided important insight into the mechanism of the F1-ATPase (4) and myosin (5, 6). It is used here to determine the rates of the reverse reactions of kinesin.Hydrolysis of ATP by kinesin proceeds through attack of water on the ␥ phosphoryl to yield P i that contains one water-derived oxygen and three oxygens from the nonbridge ␥ oxygens of the ATP, as indicated in Scheme 2 for hydrolysis of unenriched ATP in 100% 18 O-enriched water. If P i release vi...

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Equilibrium Studies of Kinesin-Nucleotide Intermediates

Cited by 49 publications

References 50 publications

“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain

The tethered motor domain of a kinesin-microtubule complex catalyzes reversible synthesis of bound ATP

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