ITEM METADATA RECORD
Title: Kinetics of association and dissociation of two enantiomers, NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI 980), to tubulin
Authors: Barbier, P ×
Peyrot, V
Dumortier, C
DHoore, A
Rener, GA
Engelborghs, Yves #
Issue Date: Jan-1996
Publisher: Amer chemical soc
Series Title: Biochemistry vol:35 issue:6 pages:2008-2015
Abstract: The kinetics of binding of R- and S-enantiomers were studied by the fluorescence stopped-flow technique. For the R-enantiomer, the time course of the increase in fluorescence is best fitted by a sum of two exponentials. In pseudo-first-order conditions, the first observed rate constant showed a linear concentration dependence whereas the second showed a hyperbolic one. The dissociation rate constants were determined independently by displacement experiments with 2-methoxy-5-(2,3,4-trimethoxyphenyl)2,4,6-cycloheptatrien-1-one (MTC). The two exponential phases were assumed to be due to a two-step binding mechanism: an initial binding followed by a conformational change. This is different from colchicine and MTC binding, where the two phases show a hyperbolic concentration dependence and are attributed to the parallel binding to different isoforms of tubulin [Banerjee, A., & Luduena, R. F. (1992) J. Biol. Chern. 267, 13335-13339]. R-isomer binding did not discriminate between the tubulin isoforms. The temperature dependence of all the rate constants were measured, and the entire thermodynamic reaction path was constructed. For the S-isomer, the direct fluorescence stopped-flow study showed that the signals were largely imputable to the fluorescence of the binding at low-affinity sites [Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rener, G. A., & Temple, C., Jr. (1993) Biochemistry 32, 10674-10682]. Therefore, we exploited the competition between R- and S-isomers to determine the binding kinetics of the S-isomer to the R-site. The observed rate constants for competitive binding showed a linear concentration dependence, thus allowing us to calculate the association rate constant of the S-isomer to the R-site. The kinetics of displacement of the S-isomer by MTC allowed the dissociation rate constant for the S-isomer to be determined. The binding of both enantiomers to tubulin in presence of tropolone methyl ether (analog of the colchicine C ring) was decreased, indicating the involvement of the C subsite.
ISSN: 0006-2960
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Biochemistry, Molecular and Structural Biology Section
× corresponding author
# (joint) last author

Files in This Item:

There are no files associated with this item.

Request a copy

 




All items in Lirias are protected by copyright, with all rights reserved.

© Web of science