Abstract
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) approximately 10(7) m(-1), 37 degrees 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 x 10(5) m(-1) s(-1) and 13.8 +/- 1.8 x 10(5) m(-1) s(-1) at 37 degrees 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.
Highlights
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)
The binding mechanism consists of a fast bimolecular reaction followed by at least two monomolecular rearrangements, which were characterized with stopped-flow techniques
These dynamics are normally controlled by the nucleotide content of tubulin; the heterodimer binds two molecules of guanine nucleotide: a nonexchangeable GTP molecule at the ␣-subunit, which plays a structural stability role (7), and a second exchangeable molecule bound to the -subunit, which can be either GTP or GDP
Summary
Taxol® (BristolMyers Squibb) (paclitaxel), 4,10-diacetoxy-2␣-(benzoyloxy)-5,20epoxy-1,7-dihydroxy-9-oxotax-11-en-13␣-yl(2R, 3S)-3-[(phenylcarbonyl) amino]-2-hydroxy-3-phenylpropionate; docetaxel (Taxotere®; Rhonemetastatic breast cancer, head and neck cancer, and lung cancer (3–5). Taxol arrests cell division by blocking microtubule dynamics, which is necessary for their function (6) These dynamics are normally controlled by the nucleotide content of tubulin; the heterodimer binds two molecules of guanine nucleotide: a nonexchangeable GTP molecule at the ␣-subunit, which plays a structural stability role (7), and a second exchangeable molecule bound to the -subunit, which can be either GTP or GDP. The same work demonstrated that the addition of Taxol to preformed microtubules made of pure tubulin alters their structure in less than 2 min Those results indicate that the taxoid binding site is accessible in the microtubules. 24 and 35) or difluorofluorescein (7-O-[N-(2, 7-difluoro-4Ј-fluoresceincarbonyl)-L-alanyl]Taxol; Flutax-2) The use of these probes, which retain the microtubule assembly activity, has allowed a detailed kinetic characterization of the reversible interaction of microtubules and taxoids.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
