Silence K, D'Hoore A, Engelborghs Y, Peyrot V, Briand C
Laboratory of Chemical and Biological Dynamics, Katholieke Universiteit Leuven, Belgium.
Biochemistry. 1992 Nov 17;31(45):11133-7. doi: 10.1021/bi00160a025.
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.
通过荧光停流法研究了MDL 27048与微管蛋白结合的动力学。这种结合伴随着荧光增强。时间进程可用两个指数之和来描述，假定这是由于存在两种主要的微管蛋白亚型。在伪一级条件下，观察到的速率常数以非线性方式依赖于MDL的浓度。这种浓度依赖性可以通过存在低亲和力的快速平衡，随后是初始复合物的异构化来描述。通过置换实验研究了解离动力学，其中MTC用作竞争性配体。根据温度依赖性确定了第一个结合平衡的反应焓变以及异构化正向和反向步骤的活化能。这对于两种微管蛋白亚型群体是可行的。在秋水仙碱A环的快速结合类似物3',4',5'-三甲氧基苯乙酮存在下，MDL与微管蛋白结合的动力学减慢，但不受托酚酮甲醚结合的影响，这表明MDL的结合位点与秋水仙碱有共同的A亚位点，但没有C亚位点。
