Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism

Abstract

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 M, and the maximum speed was 904 nm͞s. Pi behaved as a competitive inhibitor with K I ‫؍‬ 9 ؎ 1 mM. ADP behaved approximately as a competitive inhibitor with K I ‫؍‬ 35 ؎ 2 M. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and͞or 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.crossbridge cycle ͉ motor protein ͉ chemomechanical coupling K inesin is a motor protein that couples the free energy derived from the hydrolysis of ATP into mechanical work used to drive cellular motility. Two properties of kinesin are essential for its function. First, kinesin undergoes directed motion. It moves toward the plus-or fast-growing end of a microtubule as it transports membrane-bounded organelles toward the periphery of neurons and other cells (1) where the plus ends of microtubules are usually located (2). And second, kinesin is processive. An individual kinesin molecule can move up to several microns along a microtubule without dissociating (3). Processivity ensures that even a small vesicle with just one or two motors on its surface will spend a large fraction of its time attached to and moving along a microtubule (as opposed to diffusing in the cytoplasm).The directed and processive motion of kinesin is tightly coupled to the hydrolysis of ATP. High-precision tracking of kinesin-coated beads reveals that kinesin takes 8-nm steps (4) from one tubulin dimer to the next along a path that is parallel to the axis of the microtubule (5). Direct measurement of the ATPase rate and its correlation to the speed of movement indicates that in standard motility assays where the load is low, kinesin hydrolyses only one ATP per 8-nm step (6). A stoichiometry of 1 step per ATP implies that each cycle of ATP hydrolysis [the binding of ATP to kinesin's nucleotide-binding pocket, its hydrolysi...

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

“…In the first, strain weakens the microtubule-binding affinity of the rearward head, promoting P i release and detachment (the 'rear-gated head' model) [28,29]. In the second, strain prevents ATP binding to the forward head until the rearward head detaches (the 'front-gated head' model) [24,[30][31][32].…”

Microtubule transport, concentration and alignment in enclosed microfluidic channels

“…7, was 738 ± 62 nm/s (mean ± SD, N = 25) shortly after introducing the microtubules and 702 ± 58 nm/s after 90 min. Data from Schief et al (2004), who measured the gliding velocity of conventional kinesin at a range of ATP and ADP concentrations, indicate that with an initial concentration of 10 mM ATP, conversion of ATP to ADP will cause a proportional decrease in the gliding velocity. This small reduction after 90 min of operation implies there is a ∼ 5% decrease in ATP from 10 mM to 9.5 mM.…”

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

“…For example, the dimeric kinesin-1 transports cargo by taking 8 nm steps in a hand-over-hand fashion along microtubules (2)(3)(4)(5). Microtubules consist of circularly arranged protein chains, socalled protofilaments, assembled of a/b-tubulin dimers (1).…”