MECHANISM OF TUBULIN-COLCHICINE RECOGNITION - A KINETIC-STUDY OF THE BINDING OF THE COLCHICINE ANALOGS COLCHICIDE AND ISOCOLCHICINE

Colchicide (IDE) is a colchicine (COL) analogue in which the C-10 meth oxy group is replaced by a hydrogen atom. Its binding to tubulin is ac companied by a quenching of the protein fluorescence. The fluorescence decrease shows a monoexponential time dependence. The observed rate c onstant increases in a nonlinear way with the total concentration of I DE, allowing the determination of a binding constant for an initial bi nding site (K-1 = 5300 +/- 300 M-I) and the rate constant for the subs equent isomerization (k(2) = 0.071 +/- 0.002 s(-1)) at 25 degrees C. T he rate constant, k(-2), for the reversed isomerization can be determi ned by displacement experiments, Despite the minor alteration of the C -ring substituent, the kinetic and thermodynamic parameters of binding are substantially different from those of COL itself, for both steps. In isocolchicine (ISO) the carbonyl oxygen atom and the methoxy group s of the C-ring have been interchanged. Its binding to tubulin only re sults in small fluorescence and absorbance changes. Therefore competit ion experiments with MTC ,4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1 -one] were performed. ISO competes rapidly and with low affinity with MTC. Fluorimetric titrations of tubulin with MDL (MDL 27048 or trans-1 -(2,5 [4-(dimethylamino)phenyl]-2-methyl-2-propen-1-one) in the presen ce and absence of ISO give evidence for the existence of a second, slo w-reacting low-affinity site for ISO that is not accessible to MTC or MDL. The relevance of these results for the recognition of COL is anal ysed.