Analysis of positional isotope exchange in ATP by cleavage of the .beta.P-O.gamma.P bond. Demonstration of negligible positional isotope exchange by myosin

Dale, Marsha P.; Hackney, David D.

doi:10.1021/bi00399a051

Cited by 20 publications

(17 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…(III) The recovery stroke brings the lever arm ''up'' into the prepower stroke orientation, while closing the ATP binding site, thereby making the ATPase catalytically active. (IV) ATP hydrolysis remains reversible until myosin binds strongly to actin (29). The lever arm then rows the myosin fibril along the actin filament while irreversibly releasing phosphate and ADP.…”

Section: Resultsmentioning

confidence: 99%

Structural mechanism of the recovery stroke in the Myosin molecular motor

Fischer

Windshügel

Horák

et al. 2005

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

171

227

View full text Add to dashboard Cite

The power stroke pulling myosin along actin filaments during muscle contraction is achieved by a large rotation (Ϸ60°) of the myosin lever arm after ATP hydrolysis. Upon binding the next ATP, myosin dissociates from actin, but its ATPase site is still partially open and catalytically off. Myosin must then close and activate its ATPase site while returning the lever arm for the next power stroke. A mechanism for this coupling between the ATPase site and the distant lever arm is determined here by generating a continuous series of optimized intermediates between the crystallographic end-states of the recovery stroke. This yields a detailed structural model for communication between the catalytic and the force-generating regions that is consistent with experimental observations. The coupling is achieved by an amplifying cascade of conformational changes along the relay helix lying between the ATPase and the domain carrying the lever arm.chemo-mechanical coupling ͉ conformational transition ͉ Conjugate Peak Refinement ͉ muscle contraction ͉ power stroke T he myosin II head is a molecular motor that transforms chemical energy derived from the hydrolysis of ATP into mechanical work. The myosin head (or cross bridge) contains the catalytic activity, but the release of hydrolysis products is inhibited unless actin is bound. Lymn and Taylor (1) first proposed the cyclic scheme for the interaction between myosin and actin that produces motion (Fig. 1A). Underlying this cycle is myosin's ability to couple small changes in its catalytic ATPase site to large conformational changes in both the actin-binding and the distant force-generating domains with well defined communication mechanisms, which ensure that these changes are correlated so as to efficiently produce mechanical work. For instance, the communication mechanism between the ATP and actinbinding regions has been illuminated recently by crystal structures of the unconventional myosin V (2, 3). Here, we focus on one of the other essential communication pathways, the one for passing structural information between the ATPase site and the distant force-generating domain during the recovery stroke of the contractile cycle (i.e., going from states II to III in Fig. 1 A).The presence of that coupling first becomes apparent when comparing the crystallographic structures of the two end-states of the recovery-stroke ( Fig. 1 B and C) (4,5). As expected, the largest difference between these structures is in the orientation of the ''converter'' domain, which carries the lever arm and which is rotated by Ϸ60°relative to the rest of the head (referred to hereafter as the ''main body''). The other significant difference is in the ATP binding site, which is partially open before the recovery stroke, rendering the ATPase catalytically inactive (for example, see figure 6a in ref. 6). In contrast, after the recovery stroke, the ATP site is fully closed and the ␥-phosphate (␥P) group of the ATP forms an additional hydrogen bond with the amide of Gly-457 (Dictyostelium discoideum numberi...

show abstract

Section: Resultsmentioning

confidence: 99%

Structural mechanism of the recovery stroke in the Myosin molecular motor

Fischer

Windshügel

Horák

et al. 2005

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

171

227

View full text Add to dashboard Cite

show abstract

“…Intermediate exchange was analyzed by hydrolysis of 1 mM unenriched ATP in 75-80% [ 18 O]water with pyruvate kinase and 4 mM phosphoenolpyruvate to regenerate ATP. Reactions were quenched with HCl after Ϸ90% consumption of phosphoenolpyruvate and the distribution of P i species containing 0, 1, 2, 3, or 4 18 O-oxygens per P i was determined by gas chromatography͞mass spectrometry after conversion of the P i to volatile triethyl P i , essentially as described (13). Typical data and analysis for the intermediate exchange of DKH346 with and without MTs in 18 O-water are given in Table 2 (see Supporting Text and Table 2, which are published as supporting information on the PNAS web site).…”

Section: Methodsmentioning

confidence: 99%

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

Hackney

2005

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

Self Cite

View full text Add to dashboard Cite

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...

show abstract

“…4) provides insight into the structural basis of ATP hydrolysis and the role the conformational change plays in the generation of movement. It is well established that hydrolysis of ATP occurs by attack of a water molecule on the ␥-phosphorus (32)(33)(34). Examination of the ␥-phosphate pocket in both MgADP⅐BeF x ⅐ S1Dc and MgADP⅐VO 4 ⅐S1Dc does not reveal any amino acid side chains within 5.5 Å of the beryllium or vanadium that might function as a catalytic base.…”

Section: Minireview: Myosin Active Site 15851mentioning

confidence: 96%

The Structural Basis of the Myosin ATPase Activity

Rayment¹

1996

Journal of Biological Chemistry

124

View full text Add to dashboard Cite

Myosin is an ATPase that converts chemical energy into directed movement and can be viewed as a molecular motor. This protein comes in many shapes and sizes. Over 11 classes of myosin have been identified, and it is anticipated that more will be found as the search continues (2). Indeed it has been recognized in one form or another in every eukaryotic cell examined. The common feature of all of these molecules is a section close to the N terminus that can be identified as a motor domain.Over the years considerable effort has been devoted to determining the chemical and physical basis of the energy conversion by myosin. All of the isoforms examined so far exhibit similar kinetic strategies and share common features of the cycle that converts chemical energy into directed movement. The key features of this process were identified 25 years ago by Lymn and Taylor (3). Contrary to initial expectations, ATP hydrolysis in myosin is not coincident with the force-generating step (3, 4). Instead, ATP binding initially reduces the affinity of myosin for actin, after which hydrolysis of ATP occurs rapidly and results in a metastable ternary complex between myosin, ADP, and inorganic phosphate (P i ). In this state, the equilibrium complex between ATP and ADP•P i is between 1 and 10 for skeletal muscle (5-7). During this time there is rapid interconversion between substrate and products (5,8). Release of the hydrolysis products from myosin is catalyzed by the rebinding of myosin to actin. The energy transduction step occurs during product release (4).Thus myosin is an unusual enzyme in that the chemical step occurs at a different point in the contractile cycle from the energy transduction event. This most likely arose because myosin spends a very small time attached to actin (9). This feature presents several interesting biochemical problems. How is the hydrolysis of ATP coupled to energy transduction, how does myosin catalyze the hydrolysis of ATP, and what is the physical basis of the metastable state? Insight into the structural foundation of these questions has been provided recently by the structure determinations of the motor domain of Dictyostelium discoideum myosin II complexed with several nucleotides together with the earlier studies on chicken skeletal myosin subfragment-1 (10 -14). Structure of the Myosin HeadThe structure of chicken skeletal myosin subfragment-1 revealed the overall organization of the myosin heavy chain and its two associated light chains. It is a highly asymmetric molecule (Fig. 1) where the thick part of the molecule is assembled from the heavy chain and the light chains wrap around a long ␣-helix to form an extended motif (10). The molecule is characterized by several deep clefts and pockets, one of which forms the nucleotide binding site. This was initially identified (in the absence of nucleotide) from the location of the phosphate binding loop and position of amino acid residues implicated by chemical modification (15,16). A second prominent cleft splits the myosin head into two major dom...

show abstract

Analysis of positional isotope exchange in ATP by cleavage of the .beta.P-O.gamma.P bond. Demonstration of negligible positional isotope exchange by myosin

Cited by 20 publications

References 31 publications

Structural mechanism of the recovery stroke in the Myosin molecular motor

Structural mechanism of the recovery stroke in the Myosin molecular motor

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

The Structural Basis of the Myosin ATPase Activity

Contact Info

Product

Resources

About