Kinetics of dissociation of the tubulin-colchicine complex. Complete reaction scheme and comparison to thermodynamic measurements.
The slow dissociation reaction of the tubulin-colchicine complex has been characterized in purified calf brain tubulin and microtubule protein preparations, using [3H]colchicine and fluorometric measurements. It fits to a single exponential phase, within the accuracy of these measurements. The dissociation is a kinetically unfavorable reaction, with activation energy values of 114 +/- 10 and 94 +/- 10 kJ mol-1 (purified tubulin and microtubule protein, respectively). The kinetic scheme previously proposed for the tubulin-colchicine association (Lambeir, A., and Engelborghs, Y. (1981) J. Biol. Chem. 256, 3279-3282) is: T + C K1 in equilibrium TC k2 in equilibrium k-2 (TC)' where step 1 is a fast reversible binding and step 2 is a slow conformational change, whose backward rate constant (k-2) was neglected for the association study. This kinetic scheme has now been completed to include the measurements of the rate-limiting dissociation step (k-2), and of the purified calf brain tubulin preparation. The overall binding standard free energy change, calculated from the kinetic measurements, is -42.0 +/- 0.1 kJ mol-1 (fast phase of binding in 10 mM sodium phosphate buffer, 0.1 mM GTP, pH 7.0, at 37 degrees C). The binding is exothermic and the calculated enthalpy change is -26 +/- 13 kJ mol-1, which coincides with the recently determined calorimetric enthalpy value, -21 +/- 2 kJ mol-1 (Menendez, M., Laynez, J., Medrano, F. J., and Andreu, J. M. (1989) J. Biol. Chem. 264, 16367-16371), suggesting that the kinetic scheme and measurements are essentially correct.
Fernando Díaz
J
J
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Andreu
J M
JM
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002850:Chromatography, Gel; D003078:Colchicine / Q000737:chemistry*; D007700:Kinetics; D013696:Temperature; D013816:Thermodynamics; D014404:Tubulin / Q000737:chemistry*
D014404:Tubulin; D003078:Colchicine