STRUCTURES OF THYMIDYLATE SYNTHASE WITH A C-TERMINAL DELETION - ROLE OF THE C-TERMINUS IN ALIGNMENT OF 2'-DEOXYURIDINE 5'-MONOPHOSPHATE AND 5,10-METHYLENETETRAHYDROFOLATE
STRUCTURES OF THYMIDYLATE SYNTHASE WITH A C-TERMINAL DELETION - ROLE OF THE C-TERMINUS IN ALIGNMENT OF 2'-DEOXYURIDINE 5'-MONOPHOSPHATE AND 5,10-METHYLENETETRAHYDROFOLATE
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DOI：
10.1021/bi00079a007
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发表时间：
1993-07-20
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：
PERRY, KM;CARRERAS, CW;STROUD, RM
胸苷酸合酶在配体结合时经历主要的构象变化，其中羧基末端显示最大的运动（约4埃）。这种从“开放”的无配体状态到“闭合”的复合构象的运动在底物的正确取向和产物形成中起着至关重要的作用。缺乏C-末端缬氨酸（V316 Am）的突变体是无活性的。描述了V316 Am及其与dUMP、FdUMP以及FdUMP和CH 2 H4-叶酸的配合物的X射线晶体结构。结构表明，配体结合在活性位点内，但与类似的野生型胸苷酸合酶结构中的模式不同。V316 Am与FdUMP和dUMP的2.7埃二元复合物结构显示，与野生型酶中的dUMP相比，核苷酸的嘧啶和核糖部分围绕3 '-羟基枢转约20度。V316 Am与辅因子CH 2 H4叶酸和底物类似物FdUMP复合的2.7埃晶体结构显示这些配体以类似于未配体酶的开放构象结合。在该三元复合物中，辅因子的咪唑烷环是开放的，并与水反应形成5-HOCH 2 H4叶酸。5-HOCH 2 H4叶酸是5-亚胺离子中间体的结构证据，这是CH 2 H4叶酸的拟定反应形式。在三个V316 Am复合物结构中观察到的改变的配体结合模式为新型TS抑制剂的设计开辟了新的途径。
Thymidylate synthase undergoes a major conformational change upon ligand binding, where the carboxyl terminus displays the largest movement (approximately 4 angstrom). This movement from an ''open'' unliganded state to the ''closed'' complexed conformation plays a crucial role in the correct orientation of substrates and in product formation. The mutant lacking the C-terminal valine (V316Am) of the enzyme is inactive. X-ray crystal structures of V316Am and its complexes with dUMP, FdUMP, and both FdUMP and CH2H4-folate are described. The structures show that ligands are bound within the active site, but in different modes than those in analogous, wild-type thymidylate synthase structures. The 2.7-angstrom binary complex structures of V316Am with FdUMP and dUMP show that the pyrimidine and ribose moieties of the nucleotides are pivoted approximately 20-degrees around the 3'-hydroxyl compared to dUMP in the wild-type enzyme. The 2.7-angstrom crystal structure of V316Am complexed with cofactor, CH2H4folate, and the substrate analog, FdUMP, shows these ligands bound in an open conformation similar to that of the unliganded enzyme. In this ternary complex, the imidazolidine ring of the cofactor is open and has reacted with water to form 5-HOCH2H4folate. 5-HOCH2H4folate is structural evidence for the 5-iminium ion intermediate, which is the proposed reactive form of CH2H4folate. The altered ligand binding modes observed in the three V316Am complex structures open new venues for the design of novel TS inhibitors.