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Monomeric kinesin head domains hydrolyze multiple ATP molecules before release from a microtubule
1Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
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 approximately 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 microM-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 approximately 100 s-1 at saturating levels of ATP. This maximum hydrolysis rate of 100 s-1 is similar to the maximum steady state ATPase rate at saturating microtubules of approximately 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 approximately 2 per DKH357 active site (superstoichiometric). The rate constant for the burst transient was approximately 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 approximately 4 ATP molecules predicted by previous steady state measurements (Jiang, W., Stock, M., Li, X., and Hackney, D. D., submitted for publication).
Insights
This study reveals that kinesin motor protein DKH357
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Motor Proteins
Background:
- Kinesin motor proteins are crucial for intracellular transport.
- Understanding the kinetics of monomeric kinesin is essential for elucidating motor function.
- DKH357 is a monomeric kinesin motor domain investigated for its ATPase activity.
Purpose of the Study:
- To investigate the transient kinetic mechanism of microtubule-stimulated ATP hydrolysis by the monomeric kinesin motor domain DKH357.
- To determine the kinetic parameters governing the interaction of DKH357 with microtubules and nucleotides.
- To explore the relationship between ATP hydrolysis, product formation, and motor-microtubule dissociation.
Main Methods:
- Transient kinetic analysis using stopped-flow spectrophotometry.
- Monitoring microtubule (MT) association and dissociation rates via turbidity changes.
- Measuring ATP hydrolysis rates and product formation bursts.
Main Results:
- DKH357 dissociation from microtubules is promoted by both ATP and ADP, with ATP being more effective at subsaturating concentrations.
- ATP hydrolysis by DKH357 exhibits a burst of product formation, with rates comparable to steady-state hydrolysis, indicating hydrolysis is partially rate-limiting.
- The dissociation rate of DKH357 from microtubules is coupled to the termination of the product formation burst.
Conclusions:
- The kinetic mechanism of monomeric kinesin DKH357 involves coupled steps of ATP hydrolysis and microtubule dissociation.
- The observed superstoichiometric burst of product formation suggests a unique kinetic pathway for DKH357.
- These findings provide insights into the regulation of kinesin motor activity at the single-molecule level.