Alternating Site Mechanism of the Kinesin ATPase

Abstract

The processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results show that the release of two molecules of ADP from dimeric kinesin (K401) after the binding of kinesin ADP to the microtubule is a sequential process leading to biphasic kinetics. The maximum rate of release of mantADP from the first motor domain of K401 or monomeric K341 is fast (300 s-1) and independent of added nucleotide. The rate of mantADP release from the second motor domain of K401 is slow in the absence of added nucleotide (0.4 s-1) and reaches a maximum rate of 300 s-1 at saturating concentrations of ATP. High concentrations of ADP stimulate mantADP release from the second head to a maximum rate of 3.8 s-1. The nonhydrolyzable analog AMP-PNP and ATP-gamma S also stimulate ADP release from the second head (maximum rate of 30 s-1), suggesting that ATP hydrolysis is not necessary to stimulate the ADP release. These experiments establish an alternating site mechanism for dimeric kinesin whereby ATP binding to one kinesin active site stimulates the release of ADP from the second site such that the reactions occurring at the active sites of the two monomer units are kept out of phase from each other by interactions between the heads. These results define the steps of the ATPase pathway that lead to the efficient coupling of ATP hydrolysis to force production in a processive reaction whereby force production in forming a tight microtubule complex by one head is coupled to the rate-limiting release of the other head from the microtubule.

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

“…This rapid release is biphasic: the first ADP is released at >200 s −1 , while the release of the second ADP is nucleotide-dependent, with ATP-stimulated release at >100 s −1 (Table 1, [16][17][18][19]). When interpreted in the context of the Rice et al neck linker model [20][21][22], these data lead to the following pathway (Figure 2, steps K1-K4).…”

X-ray Structure and Microtubule Interaction of the Motor Domain of Neurospora crassa NcKin3, a Kinesin with Unusual Processivity,

“…Conventional kinesin, the founding member of the Kinesin-1 family, is a dimeric motor protein, which moves processively along microtubules (3)(4)(5). While doing so, it hydrolyses one molecule of ATP per step in a strictly alternating way (6)(7)(8)(9)(10), suggesting that at any time, at least one head or motor domain is firmly attached to the microtubule. The hand-over-hand mechanism as a model of conventional kinesins processive movement is now wellsupported by single molecule microscopy techniques with nanometer resolution (11)(12)(13)(14)(15) and high-resolution EM of microtubule-bound motor constructs (16), although it is still a matter of debate as to how the two heads are coordinated in detail (17).…”

ATPase Kinetic Characterization and Single Molecule Behavior of Mutant Human Kinesin Motors Defective in Microtubule-Based Motility

“…Unlike many other molecular motors, such as muscle myosin, conventional kinesin is a highly processive motor and can take >100 steps along a microtubule before dissociating from the filament (9). A considerable effort has been devoted to defining the rate constants that govern the intrinsic and microtubule-stimulated ATPase cycles of conventional kinesin (10)(11)(12)(13). Both the hydrolysis step (14) and the ADP dissociation step (15) are accelerated by microtubules (10-and 1000-fold, respectively).…”