1. 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

2. Basics 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]

3. Setup Silica beads (0.5  m) 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)

4. Velocity vs. [ATP] Michaelis-Menten kinetics yielded k cat = 680 ± 31 nms -1 and K m = 62 ± 5  M.  [ATP] independent coupling ratio (# ATP hydrolysed per advance). Poisson statistics f = 1 - exp(- C) confirms that single molecules suffice to move beads. (  2 = 0.7)

5. Stepping length Advance of kinesin molecules in clear increments of 8 nm (minority of other step sizes cannot be excluded). At limiting [ATP] k cat /K m = 11 ± 1 nms -1  M -1 implying stepping rate of 1.4 ± 0.1  M -1 s -1

6. ATP per step 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  2 = 0.6) with a rate of 1.1 ± 0.1  M -1 s -1 (Two exponentials gave  2 = 1.4 with a rate of 0.8 ± 0.06  M -1 s -1 )

7. Fluctuation analysis 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.

8. Control 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.

9. Randomness 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 %).

10. Conclusion Kinesin hydrolyses only one ATP per 8 nm step. Models consistent with this result is: 1)Alternating 16-nm steps by each of the two heads. 2)Two shorter substeps of which only one is ATP-dependent and the ATP-independent substep must be at least as fast as k cat (  t substep ≈ 15 ms and beneath resolution). 3)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.