COLCHICINE BINDING TO TUBULIN MONOMERS - A MECHANISTIC STUDY

The kinetic and thermodynamic parameters for colchicine-tubulin and de acetamidocolchicine-tubulin interaction, under the condition where tub ulin is predominantly in its dissociated state (similar to 80% monomer ), have been determined. We observe that the kinetic parameters exhibi t marked change when colchicine interacts with the monomeric form of t ubulin rather than with the dimeric form of tubulin. The reaction of c olchicine with tubulin monomers is characterized by an enhanced associ ation rate which is a consequence of the lowering of activation energy . Colchicine-tubulin interaction, which is only poorly reversible, bec omes partially reversible under this condition. Differences were also noticed in the thermodynamic parameters: the reaction of colchicine wi th tubulin monomers is enthalpy driven with small positive entropy, wh ile with tubulin dimers a large positive entropy change was reported. However, no such changes in the binding parameters were observed for t he reaction involving deacetamidocolchicine (a colchicine analog devoi d 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-tubu lin interactions such as the slow association rate, high activation en ergy, 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 subu nit of tubulin.