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Microtubules accelerate ADP release by dynein
1Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Abstract:
The effects of microtubules on the phosphate-water oxygen exchange reactions catalyzed by dynein were examined in order to determine the mechanism by which microtubules activate the ATPase. Microtubules inhibited the rate of medium exchange observed during net ATP hydrolysis. Inhibition of the exchange reaction was proportional to the extent of microtubule activation of ATP turnover with no effect on the partition coefficient. These data argue that microtubules do not increase the rate of release of phosphate from dynein; rather, they increase the rate of ADP release. Microtubules markedly inhibited medium phosphate-water exchange reactions observed in the presence of ADP and Pi. With increasing concentrations of ADP, the rate of exchange increased in parallel to the dissociation of dynein from the microtubules, suggesting that only free dynein and not the microtubule-dynein complex catalyzes the exchange reaction. The rates of dynein binding to microtubules in the absence and presence of saturating ADP were 1.6 X 10(6) and 9.8 X 10(5) M-1 s-1, respectively. ADP inhibited the rate of the ATP-induced dissociation of the microtubule-dynein complex with an apparent Kd = 0.37 mM for the binding of ADP to the microtubule-dynein complex. However, the rate of dissociation of ADP from the M.D.ADP complex was quite fast (approximately 1000 s-1). These data support the postulate of a high-energy dynein-ADP intermediate and indicate that microtubules activate the dynein ATPase by enhancing the rate of ADP release.
Insights
Microtubules activate dynein ATPase by speeding up ADP release, not phosphate. This mechanism involves dynein-ADP intermediates and how microtubules influence dynein
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Motor Proteins
Background:
- Dynein is a motor protein crucial for cellular transport.
- Understanding dynein's ATPase mechanism is key to cellular function.
- Microtubules are known to regulate dynein activity.
Purpose of the Study:
- To elucidate the mechanism by which microtubules activate dynein's ATPase activity.
- To investigate the role of phosphate-water oxygen exchange in dynein-microtubule interactions.
- To determine whether microtubules affect phosphate or ADP release from dynein.
Main Methods:
- Studying phosphate-water oxygen exchange reactions catalyzed by dynein.
- Measuring ATP hydrolysis rates in the presence and absence of microtubules.
- Analyzing the effect of varying ADP concentrations on dynein-microtubule binding and dissociation.
- Quantifying binding and dissociation rates of dynein to microtubules.
Main Results:
- Microtubules inhibited the rate of medium phosphate-water exchange during net ATP hydrolysis.
- Inhibition of exchange correlated with microtubule activation of ATP turnover, without affecting the partition coefficient.
- Microtubules inhibited phosphate-water exchange in the presence of ADP and Pi.
- ADP release, not phosphate release, was identified as the rate-limiting step enhanced by microtubules.
- ADP binding to the microtubule-dynein complex was characterized, with a fast ADP dissociation rate from the dynein-ADP complex.
Conclusions:
- Microtubules activate dynein ATPase primarily by increasing the rate of ADP release.
- The findings support the existence of a high-energy dynein-ADP intermediate.
- Dynein's interaction with microtubules is crucial for regulating its enzymatic activity and power stroke.