Mechanism of Microtubule Kinesin ATPase

Abstract

A six-step mechanism is derived for the activation of kinesin K379 ATPase by microtubules. The data are fitted by the kinetic scheme [Formula see text] where T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; MtK refers to the complex of a K379 unit with the microtubule binding site. The initial binding and release steps, 1 and 6, are treated as rapid equilibria: k2 = 200 s-1, k3 = 100 s-1, k5 = 35-40 s-1, maximum steady-state rate = 25 s-1 (50 mM NaCl, 20 degrees C). k2 was obtained from the maximum rate of fluorescence enhancement with mant-ATP as substrate, k3 was obtained from the hydrolysis transient phase for ATP or mant-ATP, and k5 was obtained from the rate of decrease in fluorescence of mant-ADP in the reaction [Formula see text]. A large excess of ATP was present with the Mt to block rebinding of mant-ADP. The rate was measured as a function of microtubule concentration and extrapolated to give the maximum rate k5. The same method was used to obtain k5 for ADP by mixing K.ADP with microtubules plus excess mant-ATP. The enhancement of fluorescence for the binding of mant-ATP is followed by a decrease in fluorescence with a rate constant of 35-40 s-1. Since the decrease must occur after hydrolysis, it may be correlated with a step or steps leading to the low fluorescence MtK.D state. In the kinetic scheme, steps 4 and 5 both contribute to determining the maximum turnover rate. At higher ionic strengths or lower protein concentrations, the MtK complex is dissociated by ATP. The maximum rate is 12 +/- 2 s-1 in 50 mM NaCl; consequently, hydrolysis occurs before dissociation. The dissociation constant of MtK in the presence of ADP is twice as large as the dissociation constant in the presence of ATP and four times larger than the KM for microtubule activation. The proposed kinetic scheme, which treats the K379 units of a dimer as independent, provides a satisfactory description of the transient and steady-state properties of the system with the possible exception of results at very low substrate concentrations.

Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP

“…Interpretation of this value in terms of the rate of ATP binding to each head depends on whether the initial force is generated upon ATP binding to either head of the kinesin molecule or only when ATP molecules bind to both heads. In the former case, the rate constant for each head equals the above value, which is approximately half the value (1-3 M Ϫ1 ⅐s Ϫ1 ) obtained previously for kinesin bound to microtubules in solution (2,3). Because the load is expected to be zero in solution, the difference may indicate that the ATP binding rate decreases as the load is increased.…”

“…If the rate of ATP hydrolysis does not depend on load, the fact that the force-generating rate for each head estimated here (45 or 90 s Ϫ1 ) is smaller than the ATP hydrolysis rate in solution suggests that force is generated after the hydrolysis step. The rate of force generation is close to the ATPase cycling rate of 40-80 s Ϫ1 per kinesin molecule (20-40 s Ϫ1 per head) in solution (2)(3)(4)(5). Therefore, the rate-limiting step of the ATPase cycle also seems to control the rate of force generation.…”

“…Kinesin ATPase activity is activated more than 2,000-fold by microtubules (1)(2)(3)(4)(5). The elementary steps in the ATPase cycle are accelerated by microtubules at 20-25ЊC from 9 s Ϫ1 to 100 s Ϫ1 for ATP hydrolysis (2), from Ͼ9 s Ϫ1 to 20-50 s Ϫ1 for P i release (3), and from Ͻ0.01 s Ϫ1 to 40-300 s Ϫ1 for ADP release (5).…”

“…The biochemistry of ATP hydrolysis by kinesin in solution has been investigated using native and recombinant proteins, with or without microtubules. The ATPase rate of kinesin is very low (Ͻ0.01 s Ϫ1 ) in the absence of microtubules at room temperature and is activated more than 2,000-fold by microtubules to Ͼ20 s Ϫ1 (1)(2)(3)(4)(5). The intermediate states of the kinesin-microtubule complex are similar to those of actomyosin, that is, M⅐K, M⅐K⅐ATP, M⅐K⅐ADP⅐P i , and M⅐K⅐ADP, where M, K, and P i are microtubule, kinesin, and inorganic phosphate, respectively.…”

“…The intermediate states of the kinesin-microtubule complex are similar to those of actomyosin, that is, M⅐K, M⅐K⅐ATP, M⅐K⅐ADP⅐P i , and M⅐K⅐ADP, where M, K, and P i are microtubule, kinesin, and inorganic phosphate, respectively. The rate-limiting step of the ATPase cycle is ADP release in the absence of microtubules, and P i or ADP release in the presence of microtubules (1)(2)(3)(4)(5).…”