Kinetic Mechanism of Monomeric Non-claret Disjunctional Protein (Ncd) ATPase*
- From the Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637
Abstract
The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules.
The kinetic mechanism of the monomer motor domain, residues 335–700, satisfied a simple scheme for the binding of 2′-3′-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, 
Equation 1
where T, D, and Pi refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively, and MtN is the complex of
an Ncd motor domain with a microtubule site. Rate constantsk
1 and k
−4 are the rates of a first order step, an isomerization induced by nucleotide binding. The apparent second order rate constants
for the binding steps are 1.5 × 106 m
−1s−1 for MANT ATP and 3.5 × 106
m
−1 s−1 for MANT ADP (conditions, 50 mm NaCl, pH 6.9, 21 °C). The rate constant of the hydrolysis step (k
2) was obtained from quench flow measurements of the phosphate burst phase corrected for the contribution of the rate of product
release to the transient rate constant. The rate of phosphate dissociation was not measured; the value was assigned to account
for a steady state rate of 3 s−1. The MtN complex is dissociated by ATP at a rate of 10 s−1 based on light scattering measurements. Dissociation constants of Ncd-nucleotide complexes from microtubules increased in
the order adenosine 5′-O-(thiotriphosphate) (ATPγS) < ADP-AlF4 < ATP < ADP < ADP-vanadate. Comparison of the properties of Ncd with a monomeric kinesin K332 (Ma and Taylor (1997) J. Biol. Chem. 272, 717–723) showed a close similarity, except that the rate constants for the hydrolysis and ADP release steps and the
steady state rate are approximately 15–20 times smaller for Ncd. There are two differences that may affect the reaction pathway.
The rate of dissociation of MtN by ATP is comparable to the rate of the hydrolysis step, and N·T may dissociate in the cycle,
whereas for kinesin, dissociation occurs after hydrolysis. The rate of dissociation of MtN by ADP is larger than the rate
of ADP release from MtN·D, whereas for the microtubule-kinesin complex, the rate of dissociation by ADP is smaller than the
rate of ADP release. The monomeric Mt·Ncd complex is not processive.
Footnotes
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↵* This work was supported by National Institutes of Health Grant GM 51858.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: The University of Chicago, 920 E. 58th St., Chicago, IL 60637. Tel.: 773-702-1660.
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↵1 The abbreviations used are: Ncd, non-claret disjunction, construct of Arg-335–Lys-700 amino acid residues; K332, kinesin construct of 332 amino acid residues; AMP-PNP, 5′-adenylyl-β,γ-imidodiphosphate; MANT, 2′-3′-O-(N-methylanthraniloyl; 3′-deoxy-2′-MANT ATP, 2′-O-(N-methylanthraniloyl)-3′-ATP; ATPγS, adenosine 5′-O-(3-thiotriphosphate); PIPES, 1,4-piperazinediethanesulfonic acid; Mt, microtubule; MtN, complex of an Ncd motor domain with a microtubule site.
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↵2 E. Pechatnikova and E. W. Taylor, manuscript in preparation.
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- Received June 10, 1997.
- Revision received September 8, 1997.
