NBD-isocolcemid-tubulin interaction: a novel one-step reaction involving no conformational adjustment of reactants.
Isocolcemid, a colcemid analogue in which the positions of the C-ring methoxy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that the substitution of a NBD group in the side chain of the B-ring of isocolcemid can reverse the effect of these structural alterations (at the C-ring) and the newly synthesized NBD-isocolcemid restores the lost biological activity. It inhibits microtubule assembly with an IC(50) of 12 microM and competes efficiently with [(3)H]colchicine, for binding to tubulin. NBD-isocolcemid has two binding sites on tubulin; one is characterized by fast binding, whereas the binding to the other site is slow. These two sites are independent and unrelated to each other. Colchicine and its analogues compete with NBD-isocolcemid for the slow site. Association and dissociation rate constants for the fast site, obtained from the stopped-flow measurements, are (7.37 +/- 0. 70) x 10(5) M(-1) s(-1) and 7.82 +/- 2.74 s(-1), respectively. While the interaction of colchicine and its analogues with tubulin involves two steps, NBD-isocolcemid binding to tubulin at the slow site has been found to be a one-step reaction. This is evident from the linear dependence of the observed rate constant (k(obs)) with both NBD-isocolcemid and tubulin concentrations. The interaction of NBD-isocolcemid with tubulin does not involve the conformational change of NBD-isocolcemid, as is evident from the unchanged CD spectra of the drug. The absence of enhanced GTPase activity of tubulin and the native-like protease cleavage pattern of the NBD-isocolcemid-tubulin complex suggest an unaltered conformation of tubulin upon NBD-isocolcemid binding to it as well. Implications of this on the mechanism of polymerization inhibition have been discussed.
Sengupta
S
S
Department of Biochemistry and Department of Biophysics, Bose Institute, Calcutta 700 054, India.
Banerjee
S
S
Chakrabarti
G
G
Mahapatra
P K
PK
Roy
S
S
Bhattacharyya
B
B
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D003078:Colchicine / Q000737:chemistry / Q000378:metabolism; D003703:Demecolcine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006041:Goats; D007202:Indicators and Reagents; D007536:Isomerism; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014316:Tritium; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism; D050257:Tubulin Modulators
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D014404:Tubulin; D050257:Tubulin Modulators; D014316:Tritium; C052280:NBD-colcemid; D009327:4-Chloro-7-nitrobenzofurazan; D003078:Colchicine; D003703:Demecolcine