Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 5, Pages 1183-1188Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0304369101
Keywords
crossbridge cycle; motor protein; chemomechanical coupling
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Funding
- NIAMS NIH HHS [F32 AR008558, R01 AR040593, AR40593, R37 AR040593, F32-AR08558] Funding Source: Medline
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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 muM, 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 muM. The data were compared to four-state kinetic models in which changes innucleotide 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.
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