Use of strain in a stereospecific catalytic mechanism: Crystal structures of Escherichia coli thymidylate synthase bound to FdUMP and methylenetetrahydrofolate
Use of strain in a stereospecific catalytic mechanism: Crystal structures of Escherichia coli thymidylate synthase bound to FdUMP and methylenetetrahydrofolate
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DOI：
10.1021/bi962936j
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发表时间：
1997-04-15
期刊：
影响因子：
2.9
通讯作者：
Montfort, WR
中科院分区：
文献类型：
--
作者：
Hyatt, DC;Maley, F;Montfort, WR
E.与基于机理的抑制剂5-氟-dUMP（FdUMP）和亚甲基四氢叶酸（CH 2 THF）结合的大肠杆菌胸苷酸合酶（TS）的测定结果为2.6和2.2埃标称分辨率，晶体学R因子分别为0.180和0.178，抑制剂和辅因子在两种结构中都是有序的，并且彼此之间以及与TS活性位点中的Cys 146之间显示共价连接。结构与以前的报告，这个复杂的（D。A.马修斯等人（1990）分子生物学杂志（J. Mol. Biol. 214，937-948），但在两个关键方面不同：（i）连接FdUMP和CH 2 THF的亚甲基桥旋转约60度至不同位置，和（ii）共价连接至Cys 146的FdUMP的C6的电子密度比嘧啶环中的其他原子更扩散。在前面的结构中观察到的配体排列使作者提出，必须发生配体几何结构的大的构象变化，以促进催化并在产物dTMP的甲基中产生正确的手性。新的结构表明了一种不同的产物形成机制，该机制不需要配体在催化过程中大大改变其构象，并且利用核苷酸-Cys 146硫醇加合物的不稳定性来避免深自由能阱并有助于从dUMP中提取质子。所有的中间体在提出的机制进行了建模和能量最小化的TS活性中心，所有可以容纳在本结构。文中还讨论了配体诱导的构象变化在TS机理中的作用以及Tyr 94在催化过程中作为碱基的可能性。
Two crystal structures for E. coli thymidylate synthase (TS) bound to the mechanism-based inhibitor 5-fluoro-dUMP (FdUMP) and methylenetetrahydrofolate (CH2THF) have been determined to 2.6 and 2.2 Angstrom nominal resolutions, with crystallographic R factors of 0.180 and 0.178, respectively, The inhibitor and cofactor are well ordered in both structures and display covalent links to each other and to Cys 146 in the TS active site. The structures are in general agreement with a previous report for this complex (D. A. Matthews et al. (1990) J. Mol. Biol. 214, 937-948), but differ in two key respects: (i) the methylene bridge linking FdUMP and CH2THF is rotated about 60 degrees to a different position and (ii) the electron density for C6 of FdUMP, which is covalently linked to Cys 146, is more diffuse than for the other atoms in the pyrimidine ring. The ligand arrangement observed in the previous structure led the authors to propose that a large conformational change in ligand geometry must occur in order to facilitate catalysis and yield the correct chirality in the methyl of product dTMP. The new structures suggest a different mechanism for product formation that does not require ligands to greatly alter their conformations during catalysis and which makes use of instability in the nucleotide-Cys 146 thiol adduct to avoid a deep free energy well and assist in proton abstraction from dUMP. All intermediates in the proposed mechanism were modeled and energy minimized in the TS active site, and all can be accommodated in the present structures. The role of ligand-induced conformational change in the TS mechanism and the possibility of Tyr 94 acting as a base during catalysis are also discussed.