Kinesin hydrolyses one ATP per 8-nm step

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 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
gt mean force lt 0.9 pN (lt 15 stall)
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Velocity vs. ATP

• Michaelis-Menten kinetics
• yielded kcat 680 31 nms-1 and 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)

5
Stepping length

• 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-1
  implying stepping rate of 1.4 0.1 mM-1 s-1

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ATP per step

• Exponential distribution gt 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)

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),
  its
• 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.

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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 gt 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 ).

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Conclusion

• 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.