Kinetic and thermodynamic analysis of taxol-induced polymerization of purified tubulin

Abstract

The kinetic and thermodynamic behavior of in vitro taxol-induced polymerization of purified tubulin has been studied. The assembly of tubulin initiated by taxol has a critical concentration of 0.1 mg/mL at 37 °C and consists of two consecutive pseudo first-order processes, a fast phase followed by a slow phase. The rate constants of the fast and slow phase polymerizations increase linearly with increasing tubulin concentration. This implies that the polymerization is a true pseudo first-order process. The In (1/t0.5) of polymerization for both fast and slow phases follows a linear function with ln [tubulin] fulfilling one of the criteria of condensation polymerization mechanism. From the Arrhenius plot, the temperature dependence of the rate of tubulin polymerization in the presence of taxol is biphasic. The apparent activation enthalpies for the overall polymerization reaction are 13.0 and 50.8 kcal/mol (1 cal = 4.1868 J), respectively, above and below 26 °C. The apparent activation enthalpies for the elongation reaction have also been determined. The values are 11.6 and 28.4 kcal/mol above and below 28 °C. The temperature dependence of the equilibrium constants as revealed by the van't Hoff plot is also biphasic. The standard enthalpy and entropy values are ΔH° = 7.4 and 22.5 kcal/mol above and below 30 °C, and ΔS° = 50.3 and 101.0 cal/(deg∙mol), at high and low temperatures, respectively. This suggests that the taxol-induced assembly of purified tubulin is a process driven by the effect of entropy.