The Binding Constant of ATP to Myosin S1 Fragment

Goody, Roger S.; Hofmann, W.; Mannherz, Hans Georg

doi:10.1111/j.1432-1033.1977.tb11742.x

Cited by 74 publications

(42 citation statements)

References 17 publications

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“…The three types of measurements that have been described for pure actin were repeated for the regulated actin in the presence or absence of calcium, namely the rate of formation of a complex between actin and M.ADP.Vit, the rate of release of vanadate ion by actin, and the rate of regeneration of ATPase activity. The rates of the three processes were proportional to actin concentration in the range from [10][11][12][13][14][15][16][17][18][19][20] ,uM actin. The apparent second-order rate constants were approximately equal for regulated actin plus calcium and pure actin, but the rates of the three reactions were reduced to 1/10th to 1/20th by removal ofcalcium ion.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of actomyosin ATPase by vanadate.

Goodno¹,

Taylor²

1982

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

118

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Actin-myosin subfragment-l (SF-1) or actin-heavy meromyosin is dissociated by the binding of ADP and vanadate (Vi) A recent study demonstrated the inhibition of myosin ATPase activity by vanadate (Vi) (1). The formation of an extremely stable myosin-ADP-vanadate complex (t112 of 1-2 days) provides an explanation of the mechanism of inhibition. Further studies to be published elsewhere have shown that the normal myosin-ADP complex binds vanadate weakly and undergoes a slow isomerization to form a stable complex that is a competitive inhibitorin which M indicates myosin. Considering the similarity in structure of vanadate and phosphate and that vanadate is bound stoichiometrically at or near the active site, the M-ADP-Vit complex may be a stable analogue of the myosin-ADP-phosphate intermediate, which is believed to be a key intermediate in the myosin and actomyosin ATPase mechanism (2).In the present study we have examined the reactions of vanadate with actomyosin to test the hypothesis that M-ADP-Vit is indeed an analogue of the myosin-products intermediate.This state, which is generally referred to as the M.ADP.Pi**, state is characterized by the following properties. The complex is much more wealdy bound to actin than is myosin alone or M-ADP (3, 4). The rates ofdissociation ofADP and Pi are greatly increased by forming a complex with actin, and this increase in rate accounts for the activation of myosin ATPase activity by actin. The control ofthe ATPase activity ofregulated actomyosin (AM) by calcium ion involves a change either in the association constant of M.ADP-Pi** to the actin-tropomyosin-troponin complex or in the rate of release of products from the AM.ADP.Pi** complex (5-7). The evidence to be presented here shows that the AM.ADP.Vit complex has the same properties. MATERIALS AND METHODSMyosin was prepared from rabbit back and leg muscles by the method described by Perry (8); actin was made by the procedure of Spudich and Watt (9); heavy meromyosin (HMM) and subfragment-1 (SF-1) were obtained by chymotryptic digestion ofmyosin and purified by the method ofWeeds and Taylor (10); native tropomyosin (the one-to-one complex of troponin and tropomyosin) was made by the procedure of Hitchcock (11).The myosin-ADP-vanadate complex was formed by incubation ofthe protein with 0.5 mM ADP and 1 or 2 mM vanadate for 15 min (1). Longer incubation times had no further effect on the state of the protein or the ability to quantitatively regenerate the ATPase activity. Free ADP and vanadate were removed when necessary by passage of the protein solution in pH 8.5 or pH 7.0 buffer over a 1-cm Dowex-1 X 8 column. In all experiments vanadate was added by dilution from a concentrated stock solution at pH 10 to avoid the possible formation of complex ions.Free vanadate was determined colorimetrically by reaction with 4-(2-pyridylazo)resorcinol (PAR) as described (1). The rate of color development was first order in vanadate and PAR concentrations over the ranges used in the experiments. Because protein-bound van...

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Section: Resultsmentioning

confidence: 99%

Inhibition of actomyosin ATPase by vanadate.

Goodno¹,

Taylor²

1982

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

118

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“…Comparison of the observed binding of ATP (KAB -3 x 10ll M-and AGOB = -15.6 kcal mol-'), ADP (KA = 106 M-1 and AG°=-8.2 kcal mol-1), and inorganic phosphate (KB = 670 M-1 and AGO = -3.8 kcal mol-1) gives AG'A = -11.8 kcal mol-P, AG'5 = -7.4 kcal mol-1andAGs = 3.6 kcal mol-1 (41)(42)(43) Fig. 2A) (1,44 [9] tants because of the small changes in distances in going from reactants to transition state and the relatively small force constants that can be developed by proteins (47).…”

Section: Ka'mentioning

confidence: 99%

On the attribution and additivity of binding energies

Jencks

1981

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

900

770

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It can be useful to describe the Gibbs free energy changes for the, binding to a protein of a molecule, A-B, and of its component parts, A and. B, in terms of the "intrinsic binding To reach a meaningful conclusion about this problem (and to avoid conclusions that depend on the sequence in which experiments are carried out), it is necessary to find a way of relating the observed binding of A and B to that of A-B (1-5). In one simple approach to this problem, the binding of A-B is considered to occur in two steps, in which the initial binding of the A or B moiety is followed by the binding of the rest of the molecule (Eq. 4). The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. KA

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“…The ATP binding isomerization (step 2) was practically irreversible. Indeed, it was possible to synthesize small amounts of ATP bound to myosin by addition of high concentrations of P i and ADP, showing all reactions after step 2 were at least moderately reversible (Goody et al 1977;Mannherz et al 1974;Sleep & Hutton 1980). The tight binding of ATP provides sufficient energy to dissociate actomyosin and the rapid kinetics shows that the route must pass through an AÁMÁATP ternary complex because the spontaneous dissociation of the AÁM complex is slow.…”

Section: Introductionmentioning

confidence: 99%

“…(1.1)), with skeletal myosin from the level of incorporation of 32 P i into the M Ã ÁATP state (Goody et al 1977). P i binding to the apo W501þ construct gave a 10% fluorescence quench, similar to ADP.…”

mentioning

confidence: 97%

Dynamics of actomyosin interactions in relation to the cross-bridge cycle

Zeng

Conibear

Dickens

et al. 2004

Phil. Trans. R. Soc. Lond. B

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Transient kinetic measurements of the actomyosin ATPase provided the basis of the Lymn-Taylor model for the cross-bridge cycle, which underpins current models of contraction. Following the determination of the structure of the myosin motor domain, it has been possible to introduce probes at defined sites and resolve the steps in more detail. Probes have been introduced in the Dicytostelium myosin II motor domain via three routes: (i) single tryptophan residues at strategic locations throughout the motor domain; (ii) green fluorescent protein fusions at the N and C termini; and (iii) labelled cysteine residues engineered across the actin-binding cleft. These studies are interpreted with reference to motor domain crystal structures and suggest that the tryptophan (W501) in the relay loop senses the lever arm position, which is controlled by the switch 2 open-to-closed transition at the active site. Actin has little effect on this process per se. A mechanism of product release is proposed in which actin has an indirect effect on the switch 2 and lever arm position to achieve mechanochemical coupling. Switch 1 closing appears to be a key step in the nucleotide-induced actin dissociation, while its opening is required for the subsequent activation of product release. This process has been probed with F239W and F242W substitutions in the switch 1 loop. The E706K mutation in skeletal myosin IIa is associated with a human myopathy. To simulate this disease we investigated the homologous mutation, E683K, in the Dictyostelium myosin motor domain.

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The Binding Constant of ATP to Myosin S1 Fragment

Cited by 74 publications

References 17 publications

Inhibition of actomyosin ATPase by vanadate.

Inhibition of actomyosin ATPase by vanadate.

On the attribution and additivity of binding energies

Dynamics of actomyosin interactions in relation to the cross-bridge cycle

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