Monomeric kinesin head domains hydrolyze multiple ATP molecules before release from a microtubule

Abstract

Transient kinetic analysis of microtubule-stimulated ATP hydrolysis by the monomeric kinesin motor domain DKH357 was performed to investigate the kinetic pattern of a monomer. Both ATP and ADP produced dissociation of the complex, microtubule (MT)-E, of microtubules with DKH357 at a maximum rate of ~45 s-1 as determined by decrease in turbidity. The maximum dissociation rate was independent of the KCl concentration between 25 and 200 mM. At subsaturating levels of nucleotide, ATP was more effective than ADP in dissociating DKH357 from MT-E (1.6 and 0.4 μM-1 s-1 for ATP and ADP, respectively, at 50 mM KCl). Addition of ATP to MT-E results in a burst of product formation with a maximum initial rate of ~100 s-1 at saturating levels of ATP. This maximum hydrolysis rate of 100 s-1 is similar to the maximum steady state of ATPase rate at saturating microtubules of ~70 s-1, and thus hydrolysis is at least partially rate-limiting. When the MT lattice was highly occupied with bound DKH357, the amplitude of the burst was ~2 per DKH357 active site (superstoichiometric). The rate constant for the burst transient was ~45 s-1, which is the same as the rate for dissociation of DKH357 from the microtubule and this suggests that dissociation and termination of the burst phase are coupled. The size of the burst increased with decreasing initial occupancy of the MT lattice with bound DKH357 and approached the value of ~4 ATP molecules predicted by previous steady state measurements (Jiang, W., Stock, M., Li, X., and Hackney, D. D., submitted for publication).