Schiffer C A, Clifton I J, Davisson V J, Santi D V, Stroud R M
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
Biochemistry. 1995 Dec 19;34(50):16279-87. doi: 10.1021/bi00050a007.
The crystal structure of human thymidylate synthase, a target for anti-cancer drugs, is determined to 3.0 A 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 alpha-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 8-fold reduction in kcat 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.
人胸苷酸合成酶是抗癌药物的作用靶点，其晶体结构已确定至3.0埃分辨率，并精修至晶体学残余误差为17.8%。该结构表明该酶参与了促进底物对接至活性位点的机制。此机制涉及活性位点环围绕相邻残基184和204发生约180度的扭转，并且表明在底物结合时，外部真核生物特异性环的有序排列以及活性位点的特征性闭合。已知高度保守但真核生物特异性插入的12个残基90 - 101（h117 - 128）以及156和157之间的8个残基（h146 - h153）在其他真核生物中为α螺旋结构，在这种晶体形式下，它们在蛋白质外部处于无序电子密度区域且彼此靠近。两个半胱氨酸[cys 202（h199）和213（h210）]距离足够近，可在每个亚基内形成二硫键，第三个半胱氨酸[cys 183（h180）]在其未结合配体的构象中可与活性位点半胱氨酸[cys 198（h195）]形成二硫键。人胸苷酸合成酶特有的氨基末端27个残基包含8个脯氨酸残基，也处于无序电子密度区域，并且在底物结合之前可能具有灵活性。耐药性突变Y6H使FdUMP亲和力降低4倍，dUMP反应的催化常数降低8倍。尽管与活性位点间接相连，但该结构提示了一种可能涉及结构变化的耐药机制。此结构为针对人酶未结合配体形式的基于结构的药物设计提供了独特机会。
