Fluorescence stopped-flow study of the interaction of tubulin with the antimitotic drug MDL 27048

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Abstract
The kinetics of the binding of MDL 27048 to tubulin have been studied by fluorescence stopped flow. The binding is accompanied by a fluorescence increase. The time course can be described by a sum of two exponentials, assumed to be due to the presence of two major tubulin isoforms. The observed rate constants depend in a nonlinear way on the concentration of MDL in pseudo-first-order conditions. This concentration dependence can be described by the presence of a fast equilibrium of low affinity, followed by an isomerization of the initial complex. The dissociation kinetics have been studied by displacement experiments, in which MTC was used as a competitive ligand. The reaction enthalpy change for the first binding equilibrium and the activation energies for the forward and reverse steps of the isomerization were determined from the temperature dependence. This was possible for the two tubulin isotype populations. The kinetics of the binding of MDL to tubulin are slowed down in the presence of 3',4',5'-trimethoxyacetophenone, a fast binding analog of the colchicine A-ring, but are not influenced by the binding of tropolone methyl ether, indicating that the binding site of MDL has the A-subsite in common with colchicine, but not the C-subsite.

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