Crystal structure of human thymidylate synthase: A structural mechanism for guiding substrates into the active site
Crystal structure of human thymidylate synthase: A structural mechanism for guiding substrates into the active site
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DOI：
10.1021/bi00050a007
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发表时间：
1995-12-19
期刊：
影响因子：
2.9
通讯作者：
Stroud, RM
中科院分区：
文献类型：
--
作者：
Schiffer, CA;Clifton, IJ;Stroud, RM
人胸苷酸合酶是抗癌药物的靶点，其晶体结构被确定为3.0埃分辨率，并被细化为17.8%的晶体学残差。该结构暗示了酶在促进底物与活性位点对接的机制中的作用。该机制涉及围绕相邻残基184和204枢转的活性位点环的大约180度的扭转，并且暗示外部真核生物特异性环沿着的有序化以及底物结合时活性位点的良好表征的闭合。已知在其他真核生物中，高度保守但真核生物特异性的12个残基90-101（h117-128）和156 - 157之间的8个残基（h146-h153）插入是α-螺旋，并且在这种晶体形式中在无序电子密度区域中紧密地位于蛋白质的外部。两个半胱氨酸[cys 202（h199）和213（h210）]足够接近以在每个亚基内形成二硫键，并且第三个半胱氨酸[cys 183（h180）]被定位以与活性位点半胱氨酸[cys 198（h195）]在其未配体构象中形成二硫键。氨基末端27个残基，独特的人TS，含有8个脯氨酸残基，也是在一个区域的无序电子密度，并可能是灵活的底物结合之前。耐药突变Y 6 H使FdUMP亲和力降低4倍，dUMP反应的k（cat）降低8倍。虽然间接连接到活性位点，结构表明可能涉及结构变化的耐药机制。这种结构提供了一个独特的机会，以结构为基础的药物设计，目的是在人类酶的配体形式。
The crystal structure of human thymidylate synthase, a target for anti-cancer drugs, is determined to 3.0 Angstrom resolution and refined to a crystallographic residual of 17.8%. The structure implicates the enzyme in a mechanism for facilitating the docking of substrates into the active site. This mechanism involves a twist of approximately 180 degrees of the active site loop, pivoted around the neighboring residues 184 and 204, and implicates ordering of external, eukaryote specific loops along with the well-characterized closure of the active site upon substrate binding. The highly conserved, but eukaryote-specific insertion of twelve residues 90-101 (h117-128), and of eight residues between 156 and 157 (h146-h153) are known to be a-helical in other eukaryotes, and lie close together on the outside of the protein in regions of disordered electron density in this crystal form. Two cysteines [cys 202 (h199) and 213 (h210)] are close enough to form a disulfide bond within each subunit, and a third cysteine [cys 183 (h180)] is positioned to form a disulfide bond with the active site cysteine [cys 198 (h195)] in its unliganded conformation. The amino terminal 27 residues, unique to human TS, contains 8 proline residues, is also in a region of disordered electron density, and is likely to be flexible prior to substrate binding. The drug resistance mutation, Y6H, confers a 4-fold reduction in FdUMP affinity and an 8-fold reduction in k(cat) for the dUMP reaction. Though indirectly connected to the active site, the structure suggests a mechanism of resistance that possibly involves a change in structure. This structure offers a unique opportunity for structure-based drug design aimed at the unliganded form of the human enzyme.