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

  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},
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… 

One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar.

The results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed.

Temperature Dependence of the Rotation and Hydrolysis Activities of F1-ATPase

Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30°C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive.

Phosphate release coupled to rotary motion of F1-ATPase

Atomistic molecular dynamics simulations are used to construct a first atomistic conformation of the intermediate state following the 40° substep of rotary motion, and to study the timing and molecular mechanism of inorganic phosphate (Pi) release coupled to the rotation.

Stepping Rotation of F1-ATPase with One, Two, or Three Altered Catalytic Sites That Bind ATP Only Slowly*

It appears that the presence of the slow β subunit(s) does not seriously affect other normal β subunits in the same F1-ATPase molecule and that the order of sequential catalytic events is faithfully maintained even when ATP binding to one or two of the catalytic sites is retarded.

Coupling of Rotation and Catalysis in F1-ATPase Revealed by Single-Molecule Imaging and Manipulation

F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits

It is shown that hybrid F1 containing one or two mutant β subunits with altered catalytic kinetics rotates in an asymmetric stepwise fashion, a process described as a 'sequential three-site mechanism'.

Single-molecule analysis of the rotation of F₁-ATPase under high hydrostatic pressure.

Asymmetry in the F1-ATPase and its implications for the rotational cycle.

The six steps of the F1-ATPase rotary catalytic cycle

These findings provide a structural basis for the entire F1-ATPase rotary catalysis cycle and identify a putative phosphate-releasing tunnel that indicates how ADP and phosphate releasing steps are coordinated.

Catalysis and rotation of F1 motor: Cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation

Results of ATPγS hydrolysis by the mutant F1 ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event, and this interim dwell catalytic dwell is called.



Direct observation of the rotation of F1-ATPase

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.

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

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

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.

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
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*

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.

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.