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 kinesin⅐ADP⅐P i complex that reverts to ATP before release of Pi during net hydrolysis was Ϸ0 and Ϸ2.6% in the absence and presence of microtubules (MTs), respectively. The rate of synthesis of bound ATP from free P i and the MT⅐kinesin⅐ADP complex was Ϸ1.7 M ؊1 ⅐s ؊1 (K0.5 ADP ‫؍‬ 70 M) 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 K inesin-1 is molecular motor that moves along microtubules (MTs) in a highly processive manner. Scheme 1 presents a minimal mechanism for ATP hydrolysis by the MT complex of a kinesin monomer motor domain (see refs. 1-3 for recent reviews of the ATPase mechanism and coupling to motility). ATP binds rapidly and is hydrolyzed at 100-300 s Ϫ1 . P i release occurs without a lag after hydrolysis, and thus P i release is at least as fast as hydrolysis (k 3 Ն k 2 ). ADP release is partially ratelimiting for conventional kinesin-1 and may be the principal rate limiting step for some other kinesin superfamily members.Another important aspect, however, is the equilibrium constant of each step, because it determines the free energy that is available at that step for coupling to movement against a load. The equilibrium constants can be determined if the rates in the reverse direction are known, but this is difficult because the overall equilibrium strongly favors hydrolysis. Oxygen isotopic exchange is a powerful technique for evaluation of the reversibility of the hydrolysis reaction and has provided important insight into the mechanism of the F1-ATPase (4) and myosin (5, 6). It is used here to determine the rates of the reverse reactions of kinesin.Hydrolysis of ATP by kinesin proceeds through attack of water on the ␥ phosphoryl to yield P i that contains one water-derived oxygen and three oxygens from the nonbridge ␥ oxygens of the ATP, as indicated in Scheme 2 for hydrolysis of unenriched ATP in 100% 18 O-enriched water. If P i release vi...

To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

“…Yet important questions remain. Does processive backward stepping synthesize ATP -a true cycle reversal as detected by Hackney et al [42]? Is ATP hydrolysis required?…”

“…One point of controversy is the position of the tethered head with respect to the bound head and microtubule lattice. Recent biochemical experiments have indicated that the rear ADP-containing head of kinesin (steps K1 and K6-K7) can infrequently synthesize ATP, suggesting that tight microtubule binding occurs, at least transiently [42]. However, because ATP resynthesis is energetically costly and inhibits processivity, it is possible that entry into the synthesis-competent state is also strain-regulated, limiting the probability of ATP resynthesis, which is measured to be <3% [42].…”

“…Recent biochemical experiments have indicated that the rear ADP-containing head of kinesin (steps K1 and K6-K7) can infrequently synthesize ATP, suggesting that tight microtubule binding occurs, at least transiently [42]. However, because ATP resynthesis is energetically costly and inhibits processivity, it is possible that entry into the synthesis-competent state is also strain-regulated, limiting the probability of ATP resynthesis, which is measured to be <3% [42]. Single-molecule imaging of the fluorescently labeled kinesin motor domains at low ATP levels indicates a predominantly two-heads-bound state [27].…”

Energy Conversion by Molecular Motors Coupled to Nucleotide Hydrolysis

“…The kinesin motor exhibits tight coupling, i.e., it hydrolyzes one ATP molecule per mechanical step [10]. After ATP has been hydrolyzed by one of the catalytic motor domains (or active sites), the inorganic phosphate is released rather fast, and both transitions together take of the order of 10 ms to be completed [11]. ADP is subsequently released from the catalytic domain, and this release process is also completed during about 10 ms [12].…”

A modified active Brownian dynamics model using asymmetric energy conversion and its application to the molecular motor system

“…In spite of the extensive single-molecule studies, how kinesin functions to generate the force for discrete translation on a single protofilament of the microtubule is not well understood, leaving many questions on, for instance, substeps, backsteps, and fraction of power stroke and diffusion to each step [10,[18][19][20][21][22][23].…”