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Kinesin hydrolyses one ATP per 8-nm step

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Kinesin hydrolyses one ATP per 8-nm step

Mark J. Schnitzer*† & Steven M. Block†‡

Departments of * Physics and † Molecular Biology, and ‡ Princeton Materials Institute, Princeton University, Princeton, New Jersey 08544, USA

Nature 24 Juli 1997, vol. 388, pp. 386-390

Kinesin:

Two-headed ATP driven motor protein that moves along the microtubules in discrete steps of 8 nm.

Central question:

How many molecules of ATP are consumed per step?

Method:

Using the processivity of kinesin, statistical analysis of intervals between steps at limiting ATP and studies of fluctuations in motor speed as a function of [ATP]

Silica beads (0.5 mm) with nonspecifically bound kinesin captured in an optical trap and deposited onto immobilized microtubules bound to the coverglass. Subsequent movements recorded with optical-trapping interferometry.

Low concentrations of kinesin protein ensures maximum one kinesin per bead.

Applied force: 7 fN nm-1 => mean force < 0.9 pN (< 15 % stall)

Michaelis-Menten kinetics

yielded kcat = 680 ± 31 nms-1and Km = 62 ± 5 mM.

- [ATP] independent coupling ratio (# ATP hydrolysed per advance).
Poisson statistics f = 1 - exp(-lC) confirms that single molecules suffice to move beads. (c2 = 0.7)

Advance of kinesin molecules in clear increments of 8 nm (minority of other step sizes cannot be excluded).

At limiting [ATP] kcat/Km = 11 ± 1 nms-1 mM-1implying stepping rate of 1.4 ± 0.1 mM-1 s-1

Exponential distribution => solitary, rate-limiting biochemical reaction (ATP binding) i.e. kinesin requires only one ATP per step.

If (n) ATP molecules were needed the distribution would be a convolution of n exponentials.

Data well fit by single exponential (reduced c2 = 0.6) with a rate of 1.1 ± 0.1 mM-1 s-1

(Two exponentials gave c2 = 1.4 with a rate of 0.8 ± 0.06 mM-1 s-1)

For single processive motors, fluctuations about the average speed reflect underlying enzyme stochasticity.

Randomness parameter, r, is a dimensionless measure of the temporal irregularity between steps. For motors that step a distance, d, and whose positions are functions of time, x(t), it’s:

Since both numerator and denominator increase linearly in time, their ratio approaches a constant. The reciprocal of this constant, r-1, supplies a continuous measure of the number of rate-limiting transitions per step.

Robust to sources of thermal and instrumental noise and without need to identify individual stepwise transitions.

Test of determinability of r performed with both simulated and real data.

Simulated: Stochastic staircase records and gaussian white noise.

Real: 2 mM AMP-PNP (non-hydrolysable ATP analogue) and movement of stage.

Both tests positive i.e. stepping statistics could be distinguished.

Saturating ATP value implies minimum two rate-limiting transitions per step. Biochemical pathways also predict r ≈ ½ with assumption of one hydrolysis per step.

For limiting [ATP], r rises through 1, reflecting a single rate-limiting transition once per advance (ATP binding).

Why is r > 1 at limiting [ATP]?

Not heterogeneity in ATP binding rate, bead size or stiffness of bead-kinesin linkage, futile hydrolysis, sticking or transient inactive states.

Maybe backwards movement (7 %) and/or double step (16 %).

Kinesin hydrolyses only one ATP per 8 nm step. Models consistent with this result is:

- Alternating 16-nm steps by each of the two heads.
- Two shorter substeps of which only one is ATP-dependent and the ATP-independent substep must be at least as fast as kcat (Dtsubstep ≈ 15 ms and beneath resolution).
- Alternatively the ATP-independent substep might be load dependent with a rate slowed with increasing load.
Another future challenge lies in understanding the molecular basis of kinesin movement, since the motor domain of kinesin is quite small (4.5 x 4.5 x 7.0 nm) compared to the 8 nm step.