Kinetics of association and dissociation of two enantiomers, NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI 980), to tubulin.
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. Chem. 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.
Barbier
P
P
URA-CNRS 1924, Faculté de Pharmacie, Marseille, France.
Peyrot
V
V
Dumortier
C
C
D'Hoore
A
A
Rener
G A
GA
Engelborghs
Y
Y
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D003078:Colchicine / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D011485:Protein Binding; D011719:Pyrazines / Q000737:chemistry* / Q000378:metabolism*; D013237:Stereoisomerism; D013552:Swine; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
D011719:Pyrazines; D014404:Tubulin; C047150:NSC 350386; D003078:Colchicine