Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase

@article{Yasuda2001ResolutionOD,
  title={Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase},
  author={Ryohei Yasuda and Hiroyuki Noji and Masasuke Yoshida and Kazuhiko Kinosita and Hiroyasu Itoh},
  journal={Nature},
  year={2001},
  volume={410},
  pages={898-904}
}
The enzyme F1-ATPase has been shown to be a rotary motor in which the central γ-subunit rotates inside the cylinder made of α3β3 subunits. At low ATP concentrations, the motor rotates in discrete 120° steps, consistent with sequential ATP hydrolysis on the three β-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120° step consists of roughly 90° and 30° substeps, each taking only a fraction of a millisecond. ATP binding drives the 90° substep, and the… 

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References

SHOWING 1-10 OF 53 REFERENCES

Direct observation of the rotation of F1-ATPase

TLDR
It is shown that a single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation of its motion by attaching a fluorescent actin filament to the γ-subunit as a marker, which enabled us to observe this motion directly.

F1-ATPase: a highly efficient rotary ATP machine.

A single molecule of F1-ATPase is by itself a rotary motor in which a central subunit, gamma, rotates against a surrounding stator cylinder made of alpha 3 beta 3 hexamer. Driven by the three beta

Rotation of subunits during catalysis by Escherichia coli F1-ATPase.

TLDR
The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis, and similar reactivities of unlabeled and radiolabeled beta sub units with gamma C87 upon reoxidation.

Intersubunit rotation in active F-ATPase

TLDR
An intersubunit rotation in real time in the functional enzyme F-ATPase is recorded by applying polarized absorption relaxation after photobleaching to immobilized F1 with eosin-labelled γ in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilization F1.

The structure of the central stalk in bovine F1-ATPase at 2.4 Å resolution

TLDR
The central stalk in ATP synthase is made of γ, δ and ɛ subunits in the mitochondrial enzyme, and with crystals of F1-ATPase inhibited with dicyclohexylcarbodiimide, the complete structure was revealed.

Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.

TLDR
Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates, and add to the evidence that a recent claim that the mitochondrial F1-ATPase does not show catalytic site cooperativity is invalid.

Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging.

  • K. AdachiR. Yasuda K. Kinosita
  • Chemistry, Biology
    Proceedings of the National Academy of Sciences of the United States of America
  • 2000
TLDR
Results show that the 120 degrees stepping is a genuine property of this molecular motor and that the rate of ATP binding is insensitive to the load exerted on the rotor subunit.

Catalytic Activity of the α3β3γ Complex of F1-ATPase without Noncatalytic Nucleotide Binding Site*

TLDR
The results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F 1- ATPase observed at ATP concentrations below ~300 μM.

Three conformational states of scallop myosin S1.

TLDR
Comparison of available crystal structures from different myosin isoforms and truncated constructs in either the nucleotide-free or transition states indicates that the major features within the motor domain are relatively invariant in both these states.
...