EVIDENCE FOR ALTERNATING HEAD CATALYSIS BY KINESIN DURING MICROTUBULE-STIMULATED ATP HYDROLYSIS

The N-terminal 392 amino acids of the Drosophila kinesin alpha subunit (designated DKH392) form a diner in solution that releases only one o f its two tightly bound ADP molecules on association with a microtubul e, 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-stat e ATPase rates, indicating that it is characteristic of the mechanism of microtubule-stimulated ATP hydrolysis and not the result of a fortu itous balance of rate constants. When [alpha-P-32]ATP is included in t he medium, incorporation of P-32 label into the pool of ADP that is bo und to the complex of DKH392 and microtubules occurs rapidly enough fo r the bound ADP to be an intermediate on the main pathway of ATP hydro lysis. These and other results are consistent with the half-site react ivity being a consequence of the tethering of dimeric DKH392 to the mi crotubule through one head domain, which is attached in a rigor-like m anner without bound nucleotide, whereas the other head is not attached to the microtubule and still contains a tightly bound ADP. An interme diate of this nature and the tight binding of DKH392 to microtubules i n the presence of ATP suggest a mechanism for directed motility in whi ch the head domains of dimeric DKH392 alternate in a sequential manner .