Medrano F J, Andreu J M, Gorbunoff M J, Timasheff S N
Consejo Superior de Investigaciones Cientificas, Madrid, Spain.
Biochemistry. 1989 Jun 27;28(13):5589-99. doi: 10.1021/bi00439a038.
The interactions of tubulin with colchicine analogues in which the tropolone methyl ether ring had been transformed into a p-carbomethoxybenzene have been characterized. The analogues were allocolchicine (ALLO) and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), the first being transformed colchicine and the second transformed colchicine with ring B eliminated. The binding of both analogues has been shown to be specific for the colchicine binding site on tubulin by competition with colchicine and podophyllotoxin. Both analogues bind reversibly to tubulin with the generation of ligand fluorescence. The binding of ALLO is slow, the fluorescence reaching a steady state in the same time span as colchicine; that of TCB is rapid. The displacement of ALLO by podophyllotoxin proceeds with a half-life of ca. 40 min. Binding isotherms generated from gel filtration and fluorescence measurements have shown that both analogues bind to tubulin with a stoichiometry of 1 mol of analogue/mol of alpha-beta tubulin. The equilibrium binding constants at 25 degrees C have been found to be (9.2 +/- 2.5) x 10(5) M-1 for ALLO and (1.0 +/- 0.2) X 10(5) M-1 for TCB. Binding of both analogues was accompanied by quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Both inhibited microtubule assembly in vitro, ALLO substoichiometrically, and both induced the abnormal cooperative polymerization of tubulin, which is characteristic of the tubulin-colchicine complex. Analysis in terms of the simple bifunctional ligand binding mechanism developed for colchicine [Andreu, J.M., & Timasheff, S.N. (1982) Biochemistry 21, 534-543] and comparison with the binding of the colchicine two-ring analogue, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one [Andreu, J. M., Gorbunoff, M. J., Lee, J. C., & Timasheff, S. N. (1984) Biochemistry 23, 1742-1752], have shown that transformation of the tropolone methyl ether part of colchicine into p-carbomethoxybenzene weakens the standard free energy of binding to tubulin by 1.4 +/- 0.1 kcal/mol, while elimination of ring B weakens it by 1.0 +/- 0.1 kcal/mol. The roles of rings C and B of colchicine in the thermodynamic and kinetic mechanisms of binding to tubulin were analyzed in terms of these findings.
已对微管蛋白与秋水仙碱类似物的相互作用进行了表征，其中托酚酮甲醚环已转化为对甲氧基羰基苯。这些类似物是别秋水仙碱（ALLO）和2,3,4-三甲氧基-4'-甲氧基羰基-1,1'-联苯（TCB），前者是转化后的秋水仙碱，后者是消除了B环的转化后的秋水仙碱。通过与秋水仙碱和鬼臼毒素竞争，已证明这两种类似物对微管蛋白上的秋水仙碱结合位点具有特异性结合。两种类似物均与微管蛋白可逆结合并产生配体荧光。ALLO的结合较慢，荧光在与秋水仙碱相同的时间跨度内达到稳态；TCB的结合则较快。鬼臼毒素取代ALLO的过程半衰期约为40分钟。通过凝胶过滤和荧光测量生成的结合等温线表明，两种类似物均以1摩尔类似物/摩尔α-β微管蛋白的化学计量比与微管蛋白结合。已发现25℃下ALLO的平衡结合常数为(9.2±2.5)×10⁵M⁻¹，TCB为(1.0±0.2)×10⁵M⁻¹。与秋水仙碱结合类似，两种类似物的结合均伴随着蛋白质荧光猝灭、微管蛋白远紫外圆二色性的扰动以及微管蛋白GTP酶活性的诱导。两者均在体外抑制微管组装，ALLO为亚化学计量抑制，且两者均诱导微管蛋白异常协同聚合，这是微管蛋白-秋水仙碱复合物的特征。根据为秋水仙碱开发的简单双功能配体结合机制进行分析[安德鲁，J.M.，& 蒂马舍夫，S.N.（1982年）《生物化学》21卷，534 - 543页]，并与秋水仙碱二环类似物2-甲氧基-5-(2,3,4-三甲氧基苯基)-2,4,6-环庚三烯-1-酮的结合进行比较[安德鲁，J.M.，戈尔布诺夫，M.J.，李，J.C.，& 蒂马舍夫，S.N.（1984年）《生物化学》23卷，1742 - 1752页]，结果表明秋水仙碱的托酚酮甲醚部分转化为对甲氧基羰基苯会使与微管蛋白结合的标准自由能降低1.4±0.1千卡/摩尔，而消除B环会使其降低1.0±0.1千卡/摩尔。根据这些发现分析了秋水仙碱的C环和B环在与微管蛋白结合的热力学和动力学机制中的作用。
