Barbier P, Peyrot V, Leynadier D, Andreu J M
Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Velazquez 144, 28006 Madrid, Spain.
Biochemistry. 1998 Jan 13;37(2):758-68. doi: 10.1021/bi970568t.
NSC 613862 (S)-(-) and NSC 613863 (R)-(+) are the two chiral isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3, 4-b]pyrazin-7-yl carbamate. Both compounds bind to tubulin in a region that overlaps the colchicine site. They induce formation of abnormal polymers from purified GTP-Mg-tubulin, the active assembly form of tubulin, in glycerol-free buffer with magnesium [De Ines, C., Leynadier, D., Barasoain, I., Peyrot, V., Garcia, P., Briand, C., Rener, G. A., and Temple, C., Jr. (1994) Cancer Res. 54, 75-84]. In this study, we observed that the S-isomer can promote polymerization of GDP-tubulin, the inactive assembly-incompetent form of tubulin, into nonmicrotubular structures at a critical protein concentration of 1 mg/mL (12 mM MgCl2). Neither the R-isomer nor colchicine have this ability. By electron microscopy, these tubulin polymers showed the same poorly defined filamentous structure when GDP-tubulin or GTP-Mg-tubulin were used. By HPLC measurements, we demonstrated that a dissociated GTP hydrolysis and exchange of nucleotide occurred during the isomer-induced abnormal assembly. Both isomers inhibited the Mg2+-induced tubulin self-association leading to 42 S double ring formation from GTP-Mg-tubulin or GDP-tubulin. Measurement of their binding under nonassociation conditions revealed a 3-fold decrease in the apparent equilibrium binding constant of the R-isomer to GDP-tubulin relative to GTP-Mg-tubulin. For the S-isomer, the decrease in the binding constant was less pronounced. Binding data, analyzed in terms of a system of linked conformational and association equilibria, provide evidence that the active ("straight") rather than the inactive ("curved") conformation of tubulin differentially recognizes these ligands. Whereas binding of colchicine to tubulin is well-known to induce GTP hydrolysis, this is the first case in which the interaction of a ligand with the colchicine site is shown to be sensitive to the presence of GDP or GTP at the distant nucleotide binding site.
NSC 613862（S）-（-）和NSC 613863（R）-（+）是5-氨基-2-甲基-1,2-二氢-3-苯基吡啶并[3,4-b]吡嗪-7-基氨基甲酸乙酯的两种手性异构体。这两种化合物在与秋水仙碱结合位点重叠的区域与微管蛋白结合。它们在不含甘油且含有镁的缓冲液中，能促使纯化的GTP-Mg-微管蛋白（微管蛋白的活性组装形式）形成异常聚合物[德·伊内斯，C.，莱纳迪耶，D.，巴拉索安，I.，佩罗，V.，加西亚，P.，布里安，C.，雷纳，G. A.，和坦普尔，C.，Jr.（1994年）《癌症研究》54，75 - 84]。在本研究中，我们观察到S-异构体能够在关键蛋白浓度为1 mg/mL（12 mM MgCl₂）时，促进GDP-微管蛋白（微管蛋白的无活性且无法组装的形式）聚合成非微管结构。R-异构体和秋水仙碱均无此能力。通过电子显微镜观察，当使用GDP-微管蛋白或GTP-Mg-微管蛋白时，这些微管蛋白聚合物呈现出相同的结构不清晰的丝状结构。通过高效液相色谱测量，我们证明在异构体诱导的异常组装过程中发生了GTP水解解离以及核苷酸交换。两种异构体均抑制Mg²⁺诱导的微管蛋白自组装，导致GTP-Mg-微管蛋白或GDP-微管蛋白形成42 S双环。在非结合条件下测量它们的结合情况发现，相对于GTP-Mg-微管蛋白，R-异构体与GDP-微管蛋白的表观平衡结合常数降低了3倍。对于S-异构体，结合常数的降低不太明显。根据相关构象和结合平衡系统分析结合数据，结果表明微管蛋白的活性（“直的”）而非无活性（“弯曲的”）构象对这些配体具有不同的识别能力。虽然众所周知秋水仙碱与微管蛋白的结合会诱导GTP水解，但这是首次表明配体与秋水仙碱结合位点的相互作用对远处核苷酸结合位点处GDP或GTP的存在敏感的情况。
