Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Heissler, Sarah M.; Liu, Xiong; Korn, Edward D.; Sellers, James R.

doi:10.1074/jbc.m113.485946

Cited by 9 publications

(10 citation statements)

References 45 publications

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“…The actin‐activated ADP release k ‐ AD (~5 s ‐1 ) is around 10 times faster than the k cat , making it unlikely to rate limit the catalytic cycle under physiologic conditions. In agreement with other reports on class‐2 myosins, the release of inorganic phosphate (P i ) is the kinetic step limiting the actomyosin ATPase cycle (23, 25). The fast ADP release rate compared with the k cat leads to a low duty ratio of 0.1, calculated from

d u t y r a t i o = \frac{\frac{1}{k_{‐ A D}} + \frac{1}{k_{+ 2}}}{\frac{1}{k_{c a t}}}

…”

Section: Discussionsupporting

confidence: 91%

“…the catalytic cycle under physiologic conditions. In agreement with other reports on class-2 myosins, the release of inorganic phosphate (P i ) is the kinetic step limiting the actomyosin ATPase cycle (23,25). The fast ADP release rate compared with the k cat leads to a low duty ratio of 0.1, calculated from…”

Section: Actomyosin Atpase Cyclesupporting

confidence: 91%

“…Its steady‐state ATPase activity and the actin‐activated ADP release rate constants are similar to those reported for human class‐2 nonmuscle myosins (Table 2) (16, 25, 36). In comparison, the reported values for class‐2 myosins from Acanthamoeba and Dictyostelium (15‐31 s ‐1 ) are significantly higher (Table 2) (23, 26). We speculate that alternatively spliced variants of fly nonmuscle myosin‐2 with insertions of either 8 or 40 amino acids in loop‐1 might have faster actin‐activated ADP release rates, as it has been described for human non‐muscle myosin‐2C and smooth muscle myosin‐2 splice variants (36–39).…”

Section: Discussionmentioning

confidence: 82%

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Kinetic characterization of the sole nonmuscle myosin‐2 from the model organismDrosophila melanogaster

2015

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Nonmuscle myosin-2 is the primary enzyme complex powering contractility of the F-actin cytoskeleton in the model organism Drosophila. Despite myosin's essential function in fly development and homeostasis, its kinetic features remain elusive. The purpose of this in vitro study is a detailed steady-state and presteady-state kinetic characterization of the Drosophila nonmuscle myosin-2 motor domain. Kinetic features are a slow steady-state ATPase activity, high affinities for F-actin and ADP, and a low duty ratio. Comparative analysis of the overall enzymatic signatures across the nonmuscle myosin-2 complement from model organisms indicates that the Drosophila protein resembles nonmuscle myosin-2s from metazoa rather than protozoa, though modulatory aspects of myosin motor function are distinct. Drosophila nonmuscle myosin-2 is uniquely insensitive toward blebbistatin, a commonly used myosin-2 inhibitor. An in silico modeling approach together with kinetic studies indicate that the nonconsensus amino acid Met466 in the Drosophila nonmuscle myosin-2 active-site loop switch-2 acts as blebbistatin desensitizer. Introduction of the M466I mutation sensitized the protein for blebbistatin, resulting in a half-maximal inhibitory concentration of 36.3 6 4.1 mM. Together, these data show that Drosophila nonmuscle myosin-2 is a bona fide molecular motor and establish an important link between switch-2 and blebbistatin sensitivity.-Heissler, S. M., Chinthalapudi, K., Sellers, J. R. Kinetic characterization of the sole nonmuscle myosin-2 from the model organism Drosophila melanogaster. FASEB

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d u t y r a t i o = \frac{\frac{1}{k_{‐ A D}} + \frac{1}{k_{+ 2}}}{\frac{1}{k_{c a t}}}

…”

Section: Discussionsupporting

confidence: 91%

Section: Actomyosin Atpase Cyclesupporting

confidence: 91%

Section: Discussionmentioning

confidence: 82%

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Kinetic characterization of the sole nonmuscle myosin‐2 from the model organismDrosophila melanogaster

2015

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“…Recently, phosphorylation of a Ser in an actin-binding surface loop of the motor domain was shown to reduce the steady-state ATPase activity by the allosteric reduction of the rate-limiting P i release ~ 3-fold (Fig. 2) [53, 54]. …”

Section: Regulation By Phosphorylationmentioning

confidence: 99%

Various Themes of Myosin Regulation

Heissler

Sellers

2016

Journal of Molecular Biology

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118

127

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Members of the myosin superfamily are actin-based molecular motors that are indispensable for cellular homeostasis. The vast functional and structural diversity of myosins accounts for the variety and complexity of the underlying allosteric regulatory mechanisms that determine the activation or inhibition of myosin motor activity and enable precise timing and spatial aspects of myosin function at the cellular level. This review focuses on the molecular basis of posttranslational regulation of eukaryotic myosins from different classes across species by allosteric intrinsic and extrinsic effectors. First, we highlight the impact of heavy and light chain phosphorylation. Second, we outline intramolecular regulatory mechanisms such as autoinhibition and subsequent activation. Third, we discuss diverse extramolecular allosteric mechanisms ranging from actin-linked regulatory mechanisms to myosin:cargo interactions. At last, we briefly outline the allosteric regulation of myosins with synthetic compounds.

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“…There are several methods to determine the duty ratio: (a) as the ratio of the number of actively stroking myosin heads to the total number of myosin heads, capable of interaction with actin filament (Fusi et al 2017;Harada et al 1990;Harris and Warshaw 1993;Uyeda et al 1990;Webb et al 2013), (b) as the ratio of the times that myosin spends in the strongly bound state (ts) to the total time of the actomyosin cycle (tc) (Heissler et al 2013;Sommese et al 2013), and (c) as the mole fraction of myosin in the strongly bound state in the presence of ATP (De La Cruz et al 2000;Guhathakurta et al 2017;Henn and De La Cruz 2005). The last two methods were used to determine the duty ratio of non-muscle myosin II (Heissler et al 2013), bovine cardiac myosin (Guhathakurta et al 2017), and myosin V (De La Cruz et al 2000) and VII (Henn and De La Cruz 2005). The value of the duty ratio for skeletal myosin (0.04 -0.05) was determined using the former method from the analysis of the sliding velocity of actin filament in the in vitro motility assay (Harris and Warshaw 1993;Uyeda et al 1990), and from the analysis of muscle fiber compliance at unloaded shortening and data on thick filament spacing in X-ray diffraction experiments (Fusi et al 2017).…”

Section: Discussionmentioning

confidence: 99%

CaATP prolongs strong actomyosin binding and promotes futile myosin stroke

Gargey

Nesmelova

et al. 2019

Preprint

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Calcium plays an essential role in muscle contraction, regulating actomyosin interaction by binding troponin of thin filaments. There are several buffers for calcium in muscle, and those buffers play a crucial role in the formation of the transient calcium wave in sarcomere upon muscle activation. One such calcium buffer in muscle is ATP. ATP is a fuel molecule, and the important role of MgATP in muscle is to bind myosin and supply energy for the power stroke. Myosin is not a specific ATPase, and CaATP also supports myosin ATPase activity. The concentration of CaATP in sarcomeres reaches 1% of all ATP available. Since 294 myosin molecules form a thick filament, naïve estimation gives three heads per filament with CaATP bound, instead of MgATP. We found that CaATP dissociates actomyosin slower than MgATP, thus increasing the time of the strong actomyosin binding. The rate of the basal CaATPase is faster than that of MgATPase, myosin readily produces futile stroke with CaATP. When calcium is upregulated, as in malignant hyperthermia, kinetics of myosin and actomyosin interaction with CaATP suggest that myosin CaATPase activity may contribute to observed muscle rigidity and enhanced muscle thermogenesis.

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Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Cited by 9 publications

References 45 publications

Kinetic characterization of the sole nonmuscle myosin‐2 from the model organismDrosophila melanogaster

Kinetic characterization of the sole nonmuscle myosin‐2 from the model organismDrosophila melanogaster

Various Themes of Myosin Regulation

CaATP prolongs strong actomyosin binding and promotes futile myosin stroke

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