INFLUENCE OF THE KINESIN NECK DOMAIN ON DIMERIZATION AND ATPASE KINETICS

Motor domains of kinesin were expressed that extend from the N terminu s to positions 346, 357, 365, 381, and 405 (designated DKH346-DKH405) to determine if the kinetic differences observed between monomeric DKH 340 and dimeric DKH392 (Hackney, D. D. (1994) Proc. Natl. Acad. Sci. U .S.A. 91, 6865-6869) were specific to these constructs or due to their oligomeric state, Sedimentation analysis indicated that DKH346, DKH35 7, and DKH365 are predominantly monomeric and that DKH381 and DKH405 a re predominantly dimeric at 0.01-0.03 mu M, the concentrations used fo r ATPase assays, In buffer with 25 mm KCl, all have high k(cat) values of 38-96 s(-1) at saturating microtubule (MT) levels, Monomeric DKH34 6, DKH357, and DKH365 have K-0.5(MT) values of 17, 9, and 1.4 mu M, re spectively, but the K,.,,,,, values for the dimeric species are signif icantly lower, with 0.02 and 0.14 mu M for DKH381 and DKH405, respecti vely, The three new monomers release all of their ADP on association w ith microtubules, whereas the two new dimers retain approximately half of their ADP, consistent with the half site reactivity observed previ ously with dimeric DKH392, Both the k(bi(ATPase)) (=k(cat)/K-0.5(M/T)) values for stimulation of ATPase by MTs and the k(bi(ADP)) for stimul ation of ADP release by MTs were determined in buffer containing 120 m M potassium acetate, The ratio of these rate constants (k(bi(ratio)) = k(bi(ATPase))/k(bi(ADP))) is 60-100 for the dimers, indicating hydrol ysis of many ATP molecules per productive encounter with a MT as obser ved previously for DKH392 (Hackney, D. D.(1995) Nature 377, 448-450), For the monomers, k(bi(ratio)) values of similar to 4 indicate that th ey also may hydrolyze more than one ATP molecule per encounter with a MT and that the mechanism of hydrolysis is therefore fundamentally dif ferent from that of actomyosin, DKH340 is an exception to this pattern and may undergo uncoupled ATP hydrolysis.