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J. Biol. Chem., Vol. 275, Issue 34, 26265-26276, August 25, 2000

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Molecular Recognition of Taxol by Microtubules
KINETICS AND THERMODYNAMICS OF BINDING OF FLUORESCENT TAXOL DERIVATIVES TO AN EXPOSED SITE^*

J. Fernando Díaz^§, Rik Strobe^¶, Yves Engelborghs^¶, André A. Souto^**, and José M. Andreu

From the  Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, C/Velázquez, 144, 28006 Madrid, Spain, the ^¶ Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Celestijnenlaan 200D, B-3001 Heverlee, Belgium, and  Instituto de Química Orgánica, Consejo Superior de Investigaciones Científicas, C/Juan de la Cierva 3, 28006 Madrid, Spain

We have determined the kinetic scheme and the reaction rates of binding to microtubules of two fluorescent taxoids, 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]Taxol (Flutax-1) and 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]Taxol (Flutax-2). Flutax-1 and Flutax-2 bind to microtubules with high affinity (K_a  10⁷ M¹, 37 °C). The binding mechanism consists of a fast bimolecular reaction followed by at least two monomolecular rearrangements, which were characterized with stopped-flow techniques. The kinetic constants of the bimolecular reaction were 6.10 ± 0.22 × 10⁵ M¹ s¹ and 13.8 ± 1.8 × 10⁵ M¹ s¹ at 37 °C, respectively. A second slow binding step has been measured employing the change of fluorescence anisotropy of the probe. The reversal of this reaction is the rate-limiting step of dissociation. A third step has been detected using small angle x-ray scattering and involves a 2-nm increase in the diameter of microtubules. It is suggested that the first step entails the binding of the Taxol moiety and the second a relative immobilization of the fluorescent probe. The equilibrium and some kinetic measurements required the use of stabilized cross-linked microtubules, which preserved taxoid binding. The results indicate that the Taxol binding site is directly accessible, in contrast with its location at lumen in the current model of microtubules. An alternative structural model is considered in which the binding site is located between protofilaments, accessible from the microtubule surface.

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