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Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis

Hackney, D.D.
Proceedings of the National Academy of Sciences of the United States of America 1994 v.91 no.15 pp. 6865-6869
Drosophila melanogaster, adenosinetriphosphatase, enzyme activity, adenosine triphosphate, hydrolysis, microtubules, adenosine diphosphate
The N-terminal 392 amino acids of the Drosophila kinesin alpha subunit (designated DKH392) form a dimer in solution that releases only one of its two tightly bound ADP molecules on association with a microtubule, whereas a shorter monomeric construct (designated DKH340) releases greater than or equal to 95% of its one bound ADP on association with a microtubule. This half-site reactivity of dimeric DKH392 is observed over a wide range of ratios of DKH392 to microtubules and steady-state ATPase rates, indicating that it is characteristic of the mechanism of microtubule-stimulated ATP hydrolysis and not the result of a fortuitous balance of rate constants. When [alpha-(32)P]ATP is included in the medium, incorporation of (32)P label into the pool of ADP that is bound to the complex of DKH392 and microtubules occurs rapidly enough for the bound ADP to be an intermediate on the main pathway of ATP hydrolysis. These and other results are consistent with the half-site reactivity being a consequence of the tethering of dimeric DKH392 to the microtubule through one head domain, which is attached in a rigor-like manner without bound nucleotide, whereas the other head is not attached to the microtubule and still contains a tightly bound ADP. An intermediate of this nature and the fight binding of DKH392 to microtubules in the presence of ATP suggest a mechanism for directed motility in which the head domains of dimeric DKH392 alternate in a sequential manner.