Kinetic Analysis of ATPase Mechanisms

Trentham, David R.; Eccleston, John F.; Bagshaw, Clive R.

doi:10.1017/s0033583500002419

Cited by 286 publications

(195 citation statements)

References 163 publications

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“…Atomic coordinates were obtained from the Protein Data Bank (accession number 1FMV). similar to that observed previously [42]. For the labelled S1 samples, the basal Mg 2+ ATPase activities were similar to or greater, and the actin activation lower, than for the unlabelled S1.…”

Section: Resultssupporting

confidence: 89%

Dynamic reorganization of the motor domain of myosin subfragment 1 in different nucleotide states

Bódis

Szarka

Nyitrai

et al. 2003

European Journal of Biochemistry

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, were shown to provide an excellent structural framework for using to understand the mechanism of muscle contraction. According to these D. discoideum structures, S1.ADP.BeF x resembles the S1.ATP conformation, whereas S1.ADP.AlF -4 and S1. ADP.V i resemble the S1.ADP.P i conformation. On the other hand, the smooth muscle myosin S1 atomic structures with ADP.BeF x and ADP.AlF -4 were almost identical [7]. Analysis of these atomic models revealed that a key structural part of the nucleotide induced conformational changes in the core of the motor domain is the switch 2 (SWII) element, which consists of the SWII helix (residues 475-509) and the SWII loop (residues 511-520). The SWII element can be in an open or closed conformation in the individual states of the ATPase cycle [8]. The two conformations are in a dynamic equilibrium, which is controlled by the bound nucleotide. The open state is dominant in the pre-and postpower-stroke states, such as the apo-enzyme or S1 with bound ATP or ADP, or in the nucleotide states mimicked by b-c-imidoadenosine 5¢-triphosphate or ATPcS [8]. The closed conformation was attributed to the transition state and was observed with bound ADP.P i analogues, ADP.V i or ADP.AlF -4 . In the ADP.BeF x bound motor domain, both the open and closed conformation could be detected [5,7]. During the open-to-closed transition, the SWII element moves towards the c-phosphate [8]. This transition step can be followed by the hydrolysis of ATP and the closure of the active site through the relative rotation of the 50 kDa upper domain and the 50 kDa lower domain. The helix consisting of residues 648-666 is in the fulcrum of this rotation. In conjunction with this transition, the converter domain rotates by 60°, which induces the movement of the C-terminal end of S1 by 12 nm [9].Tryptophan fluorescence has proved to be a powerful experimental tool when used to characterize the different aspects of myosin interaction with nucleotides [10][11][12][13]. Rapid kinetic experiments using tryptophan fluorescence indicated Correspondence to B. Somogyi,

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Section: Resultssupporting

confidence: 89%

Dynamic reorganization of the motor domain of myosin subfragment 1 in different nucleotide states

Bódis

Szarka

Nyitrai

et al. 2003

European Journal of Biochemistry

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“…This predicts that the rate will be proportional to the concentration of M-ADP, which is in turn equal to the total active-site concentration when stoichiometric ADP is present. During the steady-state of ATP hydrolysis, however, M-ADP drops to about 4% of the total site concentration, because kcat is 25-fold greater than the rate of ADP release (22). Under these conditions, the rate of irreversible inhibition by V1 drops to about 3%, suggesting that M-ADP is, in fact, the main binary intermediate.…”

Section: Resultsmentioning

confidence: 97%

“…A notable difference between Mt-ADP-Vi and M**-ADP-Pi is that the latter dissociates with a t 1/2 of about 12 sec, whereas the former has a t 1/2 of a day or more. Because the rate of dissociation of M**-ADP-Pi is controlled by the isomerization step M**-ADP-Pi -M*-ADP-Pi (22), it is plausible that the dissociation of Mt-ADP-Vi is controlled by the step Mt-ADP-Vi -M-ADP-Vi. Thus, the difference in rates probably derives from the ability of Vi to lock the myosin into the Mt-ADP-Vi conformation.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of myosin ATPase by vanadate ion.

Goodno¹

1979

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

274

218

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Inhibition of the myosin ATPase by vanadate ion (V1) has been studied in 90 mM NaCl/5 mM MgCl2/20 mM Tris HCI, pH 8.5, at 250C. Although the onset of inhibition during the assay is slow and dependent upon Vi concentration (kap -t 0.3 M-1 s-'), the final level of inhibition approaches iOO4o, provided the V; concentration is in slight excess over the concentration of ATPase sites. Inhibition is not reversible by dialysis or the addition of reducing agents. The source of this irreversible inhibition consists of the formation of a stable, inactive complex with the composition MADP-V1 (where M represents a single myosin active site). The complex has been isolated, and its mechanism of formation from M, ADP, and Vi has been studied. Omission of ATP increases the rate of formation by about 35-fold (kapp 11 M-1 s-'), yet this rate is still low in comparison with the rates of simple protein-ligand association reactions. This slowness is interpreted in terms of a rate-limiting isomerization step that follows the association of M, ADP, and V1: M*ADP*V1 _ Mt.ADP.V1 (t indicates the inactive product of the isomerization). (9) and Weeds and Taylor (10), respectively. The HMM fraction that precipitated between 45 and 60% saturated ammonium sulfate was dialyzed free of ammonium sulfate, centrifuged 30 min at 40,000 X g, and used within 10 days. The concentration of HMM was expressed in terms of the ATPase-site concentration, which was determined spectrophotometrically by using a value of A2801% = 6.47 (11), assuming a molecular weight of 340,000 and two ATPase sites per molecule.ATPase assays were carried out in buffer A (0.09 M NaCl/5 mM MgCl2/20 mM Tris-HCI, pH 8.5) at 250C by the addition of MgATP (final concentration, 1 mM) to HMM at 1-7 AM sites.Assay times ranged from 0.1 to 5 hr. The reaction was stopped in aliquots (1 ml) of the assay solution with 1 ml of 10% trichloroacetic acid. The aliquots were then clarified by centrifugation and half of each was analyzed for Pi by the procedure of Taussky and Shorr (12). V1 concentrations below 10 mM caused less than 1% interference.Vanadium Analysis. Stock solutions of V1 were prepared from either Na3VO4 (adjusted to pH 10 with 6 M HCl) or V205 (adjusted to pH 10 with 10 M NaOH) and then boiled to destroy yellow polymeric species such as V100286-(13). Standard solutions were prepared by volumetric dilution. In order to minimize the pH-dependent polymerization of Vi, all studies were carried out under the alkaline conditions used for the ATPase assays (buffer A). UV-visible spectra of Vi standard solutions were obtained by using a Cary 14 spectrophotometer, and the extinction coefficient was determined: Xrnax = 265 nm, C265 = 2925 M-1 cm-1. The Vi concentration was determined spectrophotometrically wherever possible.Where this was unfeasible (e.g., in the presence of protein), vanadium was determined by a modification of the colorimetric procedure of Pribil (14), using the metallochromic dye PAR.To a 1-ml sample in buffer A was added 100 Al of 1 M imidazole (pH 6.0) and ...

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“…Stopped-flow techniques with fluorescence and absorbance monitors, as well as rapid sampling for subsequent chemical or isotopic analysis, have been used to provide information about the following principal steps in the hydrolysis pathway, where M denotes a single ATPase site of myosin or its subfragments (Trentham et al, 1976;Taylor, 1979):…”

Section: Introductionmentioning

confidence: 99%

A transient kinetic study of enthalpy changes during the reaction of myosin subfragment 1 with ATP.

Millar

Howarth

Gutfreund

1987

Biochemical Journal

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1. The enthalpy changes during individual reaction steps of the myosin subfragment 1 ATPase were studied with the use of a new stopped-flow calorimeter [Howarth, Millar & Gutfreund (1987) Biochem. J. 248, 677-682]. 2. At 5 degrees C and pH 7.0, the endothermic on-enzyme ATP-cleavage step was observed directly (delta H = +64 kJ.mol-1). 3. ADP binding is accompanied by a biphasic enthalpy change. 4. The release and uptake of protons was investigated by the use of two buffers with widely different heats of ionization. 5. Protons are involved in all four principal steps of the myosin subfragment 1 ATPase.

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Kinetic Analysis of ATPase Mechanisms

Cited by 286 publications

References 163 publications

Dynamic reorganization of the motor domain of myosin subfragment 1 in different nucleotide states

Dynamic reorganization of the motor domain of myosin subfragment 1 in different nucleotide states

Inhibition of myosin ATPase by vanadate ion.

A transient kinetic study of enthalpy changes during the reaction of myosin subfragment 1 with ATP.

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