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Volume 272, Number 49, Issue of December 5, 1997 pp. 30735-30740
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Kinetic Mechanism of Monomeric Non-claret Disjunctional Protein (Ncd) ATPase

(Received for publication, June 10, 1997, and in revised form, September 8, 1997)

From the Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637

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,

(Eq. 1)

where T, D, and P_i 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 constants k₁ and k₄ 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 M¹ s¹ for MANT ATP and 3.5 × 10⁶ M¹ s¹ for MANT ADP (conditions, 50 mM NaCl, pH 6.9, 21 °C). The rate constant of the hydrolysis step (k₂) 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 s¹. The MtN complex is dissociated by ATP at a rate of 10 s¹ based on light scattering measurements. Dissociation constants of Ncd-nucleotide complexes from microtubules increased in the order adenosine 5-O-(thiotriphosphate) (ATPS) < 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.

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