Evidence for an alternative pathway for colchicine binding to tubulin, based on the binding kinetics of the constituent rings.

The kinetics of tropolone methyl ether binding to tubulin were measured by following the loss of colchicine binding capacity upon preincubation of tubulin with tropolone methyl ether. At 25 degrees C a bimolecular association rate constant of 2.7 (+/- 0.2) M-1 min-1 was determined, and from the temperature dependence an activation energy of 37 (+/- 8) kJ.mol-1 was calculated. By displacement experiments a dissociation rate constant of 2.9 (+/- 0.6) x 10(-2) min-1 was determined at 25 degrees C. The effect of 3',4',5'-trimethoxyacetophenone (TMA) is 2-fold. TMA reduces the apparent association rate constant of colchicine, indicating that it equilibrates very rapidly and reversibly with the colchicine binding site. From this reduction the binding constant for TMA can be obtained. At 25 degrees C a value of 112 (+/- 13) M-1 is estimated. The binding of TMA is practically thermoneutral. Preincubation of tubulin with TMA over 30 min not only reduces the subsequent binding rate constant of colchicine but also the amplitude. This indicates that TMA also binds slowly in a second mode or site. Stopped-flow kinetic studies reveal that fast TMA binding competes for the initial binding of colchicine. From these results it is concluded that colchicine binds initially with its trimethoxybenzene ring and in a subsequent step with the tropolone ring.