INTERACTING HEAD MECHANISM OF MICROTUBULE-KINESIN ATPASE

Kinetic and equilibrium properties are compared for a monomeric kinesi n construct (K332) and a dimeric construct (K379), MtK379 has a low af finity (5 x 10(4) M(-1)) and a high affinity (5 x 10(6) M(-1)) binding site for mant ADP while MtK332 has a single low affinity site (5 x 10 (4) M-1). Rate constants of dissociation of mant ADP are <1 s(-1) for the high affinity site and 75-100 s(-1) for the low affinity site for MtK379. For MtK332, the effective rate constant is 200-300 s(-1). It i s proposed that the two heads of the dimer are different through the i nteraction with the microtubule, a strongly bound head with low affini ty for 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mant ADP), similar to the single strongly bound head of the monomer and a weakly bound or detached head with high affinity for mant ADP. Rate of binding of mant ADP gave an ''S''-shaped dependence on concentration for MtK379 and a hyperbolic dependence for MtK332. Binding of K379 man t ADP dimer to microtubules releases only one mant ADP at a rate of 50 s(-1). The second strongly bound mant ADP is released by binding of n ucleotides to the other head. Rates are 100 s(-1) for ATP, 30 s(-1) fo r AMPPNP or ATP gamma S, and 2 s(-1) for ADP, The rate of binding of m ant ATP to MtK379 showed an ''S''-shaped concentration dependence and limiting rate at zero concentration is <1 s-1 while MtK332 gave a hype rbolic dependence and limiting rate of 100 s(-1). The limiting rate is determined by the rate of dissociation of mant ADP in the hydrolysis cycle. The evidence is consistent with an interacting site model in wh ich binding of ATP to one head is required for release of ADP from the other head in the hydrolysis cycle. This model, in which the cycles a re maintained partly out of phase, is an extension of the alternating site model of Hackney (Hackney, D. D. (1994) Proc. Nat. Acad. Sci. U. S. A. 91, 6865-6869). It provides a basis for a processive mechanism.