KINETIC MECHANISM OF MONOMERIC NON-CLARET DISJUNCTIONAL PROTEIN (NCD)ATPASE

The non-claret disjunctional protein (Ncd) is a kinesin-related microt ubule motor that moves toward the negative end of microtubules. The ki netic mechanism of the monomer motor domain, residues 335-700, satisfi ed a simple scheme for the binding of 2'-3'-O-(N-methylanthraniloyl) ( MANT) ATP, the hydrolysis step, and the binding and release of MANT AD P, [GRAPHICS] where T, D, and P-i refer to nucleotide triphosphate, nu cleotide diphosphate, and inorganic phosphate, respectively, and MtN i s the complex of an Ncd motor domain with a microtubule site, Rate con stants k(1) and k(-4) are the rates of a first order step, an isomeriz ation induced by nucleotide binding, The apparent second order rate co nstants for the binding steps are 1.5 x 10(6) M-1 s(-1) for MANT ATP a nd 3.5 x 10(6) M-1 s(-1) for MANT ADP (conditions, 50 mM NaCl, pH 6.9, 21 degrees C), The rate constant of the hydrolysis step (k(2)) was ob tained from quench flow measurements of the phosphate burst phase corr ected for the contribution of the rate of product release to the trans ient rate constant, The rate of phosphate dissociation was not measure d; 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 o n light scattering measurements, Dissociation constants of Ncd-nucleot ide complexes from microtubules increased in the order adenosine 5'-O- (thiotriphosphate) (ATP gamma S) < ADP-AlF4 < ATP < ADP < ADP-vanadate , Comparison of the properties of Ncd with a monomeric kinesin K332 (M a and Taylor (1997) J. Biol. Chem, 272, 717-723) showed a close simila rity, except that the rate constants for the hydrolysis and ADP re lea se steps and the steady state rate are approximately 15-20 times small er for Ncd, There are two differences that may affect the reaction pat hway, 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, wherea s for kinesin, dissociation occurs after hydrolysis, The rate of disso ciation of MtN by ADP is larger than the rate of ADP release from MtN . D, whereas for the microtubule-kinesin complex, the rate of dissocia tion by ADP is smaller than the rate of ADP release, The monomeric Mt . Ncd complex is not processive.