The tethered motor domain of a kinesin-microtubule complex catalyzes reversible synthesis of bound ATP

Abstract

Although the steps for the forward reaction of ATP hydrolysis by the motor protein kinesin have been studied extensively, the rates for the reverse reactions and thus the energy changes at each step are not as well defined. Oxygen isotopic exchange between water and P_i was used to evaluate the reverse rates. The fraction of the kinesin·ADP·P_i complex that reverts to ATP before release of P_i during net hydrolysis was ≈0 and ≈2.6% in the absence and presence of microtubules (MTs), respectively. The rate of synthesis of bound ATP from free P_i and the MT·kinesin·ADP complex was ≈1.7 M^–1·s^–1 (K _0.5 ADP = 70 μM) with monomeric kinesin in the absence of net hydrolysis. Synthesis of bound ATP from the ADP of the tethered head of a dimer–MT complex was 20-fold faster than for the monomer–MT complex. This MT-activated ATP synthesis at the tethered head is in marked contrast to the lack of MT stimulation of ADP release from the same site. The more rapid ATP synthesis with dimers suggests that the tethered head binds behind the strongly attached head, because this positions the neck linker of the tethered head toward the plus end of the MT and would thus facilitate its docking on synthesis of ATP. The observed rate of ATP synthesis also puts limits on the overall energetics that suggest that a significant fraction of the free energy of ATP hydrolysis is available to drive the docking of the neck linker on binding of ATP.

• ATPase
• energy coupling
• motility
• motor protein
• isotopic exchange