4 research outputs found
Colchicine binding to tubulin monomers: a mechanistic study
The kinetic and thermodynamic parameters for colchicine-tubulin and deacetamidocolchicine-tubulin interaction, under the condition where tubulin is predominantly in its dissociated state (~80% monomer), have been determined. We observe that the kinetic parameters exihibit marked change when colchicine interacts with the monomeric form of tubulin rather than with the dimeric form of tubulin. The reaction of colchicine with tubulin monomers is characterized by an enhanced association rate which is a consequence of the lowering of activation energy. Colchicine-tubulin interaction, which is only poorly reversible, becomes partially reversible under this condition. Differences were also noticed in the thermodynamic parameters: the reaction of colchicine with tubulin monomers is enthalpy driven with small positive entropy, while with tubulin dimers a large positive entropy change was reported. However, no such changes in the binding parameters were observed for the reaction involving deacetamidocolchicine (a colchicine analog devoid of a side chain at the C-7 position of B-ring) with tubulin monomers. We therefore conclude that a single subunit of tubulin is capable of binding colchicine and that the unusual properties of colchicine-tubulin interactions such as the slow association rate, high activation energy, and the poor reversibility are due to the possible contact(s) of the C-7 substituent (in the B-ring) of colchicine with the other subunit of tubulin
Recommended from our members
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 IC50 of 12 μM and competes efficiently with [3H]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) × 105 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 bindingto 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 (kobs) 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