Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism

Proc Natl Acad Sci U S A. 2004 Feb 3;101(5):1183-8. doi: 10.1073/pnas.0304369101. Epub 2004 Jan 20.


The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the K(M) (the [ATP] required for half-maximal speed) was 28 +/- 1 microM, and the maximum speed was 904 nm/s. P(i) behaved as a competitive inhibitor with K(I) = 9 +/- 1 mM. ADP behaved approximately as a competitive inhibitor with K(I) = 35 +/- 2 microM. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and/or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P(i) is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P(i) before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P(i) release from the other.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Diphosphate / pharmacology*
  • Kinesin / antagonists & inhibitors*
  • Kinetics
  • Models, Theoretical
  • Phosphates / pharmacology*


  • Phosphates
  • Adenosine Diphosphate
  • Kinesin