Cryoenzymic studies on myosin: transient kinetic evidence for two types of head with different ATP binding properties

Tesi, Chiara; Bachouchi, Nacima; Barman, Thomas E.; Travers, Franck

doi:10.1016/0300-9084(89)90008-4

Cited by 14 publications

(14 citation statements)

References 40 publications

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“…As found previously [for acto-S 1, see Biosca et al (1985); for SI, see Tesi et al (1989); for myofibrils, see Houadjeto et al (1992)], some of the progress curves were biphasic (e.g., with acto-S 1 in Figure 4). This kinetic heterogeneity can be explained by the interference of a second site that binds ATP loosely, possibly without hydrolysis (Tesi et al, 1989). In cases, e.g., Figure 4, where the data are clearly biphasic, two exponentials were used to fit the complete set of inhibition data.…”

Section: Methodssupporting

confidence: 75%

Inhibition of ATP Binding to Myofibrils and Acto-Myosin Subfragment 1 by Caged ATP

Sleep¹,

Herrmann²,

Barman³

et al. 1994

Biochemistry

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The inhibitory effect of P3-[1-(2-nitrophenyl)ethyl]adenosine 5'-triphosphate (caged ATP) on the binding of Mg2+-ATP to myofibrils was investigated. The most sensitive method was found to be the monitoring of single turnovers of [gamma-32P] ATP hydrolysis using the quench flow technique. The method was tested using ADP, which was found to have an inhibition constant of 145 microM, in agreement with previous reports. Caged ATP behaved as a simple competitive inhibitor of ATP binding with an inhibition constant of 1.6 mM. The inhibitory effect of these ligands on the binding of ATP to acto-myosin subfragment 1 was investigated using the same method. The inhibition constants of caged ATP and ADP were found to be 0.35 mM and 50 microM, respectively. This inhibitory effect of caged ATP on ATP binding accounts for the lower rate of ATP binding to fibers, deduced from caged ATP [(0.5-1) x 10(6) M-1 s-1], than that reported for acto-S1 (3.5 x 10(6) M-1 s-1) [Goldman, Y. E., Hibberd, M. G., & Trentham, D. R. (1984) J. Physiol. (London) 354, 577].

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Section: Methodssupporting

confidence: 75%

Inhibition of ATP Binding to Myofibrils and Acto-Myosin Subfragment 1 by Caged ATP

Sleep¹,

Herrmann²,

Barman³

et al. 1994

Biochemistry

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“…S8) or other changes in the fitting procedure (Table S1). Instead, a slow phase emerged, consistent with basal ATP turnover 10,19,40 . The latter phase also emerged with addition of TX/TQ alone, whereas the fastest phase was not fully removed in this case ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 66%

Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms

et al. 2021

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Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2–0.5 s−1) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction.

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“…The calcium dependent kinetic mechanism of the myofibril and regulated actomyosin ATPase have been extensively studied 33, 39, 40, 66–68 . While the rate constants for ATP binding and ADP dissociation for unloaded myofibrils is approximately that of regulated acto-S1, they showed no significant calcium dependence.…”

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“…The ATPase cycle of actomyosin consists of the following steps: the equilibrium of the initial actomyosin–ATP complex (constant K 1 ), the myosin head isomerization step ( k 2 ′), the ATP hydrolysis step ( k 3 ), phosphate release ( k 4 ′), and ADP release ( k 5 ′) (Scheme ). The calcium-dependent kinetic mechanism of the myofibril and regulated actomyosin ATPase have been extensively studied. ,,,− While the rate constants for ATP binding and ADP dissociation for unloaded myofibrils are approximately that of regulated acto-S1, they showed no significant calcium dependence. However, the rate constant for the phosphate burst transient is larger in the presence of calcium than in the absence of calcium, which is also in agreement with regulated acto-S1. , These studies suggest that ATP binding and ADP dissociation events are not regulated by calcium.…”

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Effect of N-Terminal Extension of Cardiac Troponin I on the Ca²⁺ Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils

et al. 2016

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Cardiac troponin I (cTnI) has a unique N-terminal extension that plays a role in modifying the calcium regulation of cardiac muscle contraction. Restrictive cleavage of the N-terminal extension of cTnI occurs under stress conditions as a physiological adaptation. Recent studies have shown that in comparison with controls, transgenic mouse cardiac myofibrils containing cTnI lacking the N-terminal extension (cTnI-ND) had lower sensitivity to calcium activation of ATPase, resulting in enhanced ventricular relaxation and cardiac function. To investigate which step(s) of the ATPase cycle is regulated by the N-terminal extension of cTnI, here we studied the calcium dependence of cardiac myosin II ATPase kinetics in isolated cardiac myofibrils. ATP binding and ADP dissociation rates were measured by using stopped flow spectrofluorimetry with mant-dATP and mant-dADP, respectively. We found that the second order mant-dATP binding rate of cTnI-ND mouse cardiac myofibrils was three-fold as fast as that of wild type myofibrils in low Ca2+. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentrations while the wild type controls were not significantly affected. These data from experiments using native cardiac myofibrils under physiological conditions indicate that modification of the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase.

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Cryoenzymic studies on myosin: transient kinetic evidence for two types of head with different ATP binding properties

Cited by 14 publications

References 40 publications

Inhibition of ATP Binding to Myofibrils and Acto-Myosin Subfragment 1 by Caged ATP

Inhibition of ATP Binding to Myofibrils and Acto-Myosin Subfragment 1 by Caged ATP

Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms

Effect of N-Terminal Extension of Cardiac Troponin I on the Ca²⁺ Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils

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Cryoenzymic studies on myosin: transient kinetic evidence for two types of head with different ATP binding properties

Cited by 14 publications

References 40 publications

Inhibition of ATP Binding to Myofibrils and Acto-Myosin Subfragment 1 by Caged ATP

Inhibition of ATP Binding to Myofibrils and Acto-Myosin Subfragment 1 by Caged ATP

Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms

Effect of N-Terminal Extension of Cardiac Troponin I on the Ca2+ Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils

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Effect of N-Terminal Extension of Cardiac Troponin I on the Ca²⁺ Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils