Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution.
Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution.
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溶液中人二氢叶酸还原酶的序列特异性 1H 和 15N 共振分配。
DOI：
10.1021/bi00116a031
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Freisheim,JH
中科院分区：
文献类型：
--
作者：
Stockman,BJ;Nirmala,NR;Wagner,G;Delcamp,TJ;DeYarman,MT;Freisheim,JH
哈佛医学院生物化学与分子药理学系，马萨诸塞州波士顿02115；俄亥俄医学院生物化学与分子生物学系，俄亥俄州托莱多43699摘要：二氢叶酸还原酶是叶酸拮抗剂的细胞内靶酶，包括抗癌药物甲氨蝶呤。为了设计具有改变结合特性的新药，需要对溶液中蛋白质-药物相互作用的详细描述，以了解药物结合的特异性。作为这一过程的第一步，异核三维核磁共振波谱已被用于对超过90%的人类二氢叶酸还原酶与甲氨蝶呤络合的残基进行顺序共振分配。由于同核二维谱不能提供足够的共振色散，因此需要用15N均匀富集21.5 kda蛋白，才能通过异核三维核磁共振波谱获得共振分配。中、远程NOE被用来表征溶液中二元配体-酶配合物的二级结构。二氢叶酸还原酶（5,6,7,8 -四氢叶酸：NADP+氧化还原酶，EC 1.5）1.3)催化nadph依赖的7,8 -二氢叶酸还原为5,6,7,8 -四氢叶酸。四氢叶酸或其衍生物是嘌呤核苷酸、胸苷酸和几种氨基酸的生物合成中必不可少的辅助因子。不能维持足量的四环素
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43699 Received July 17, 1991; Revised Manuscript Received September 25, 1991 abstract: Dihydrofolate reductase is an intracellular target enzyme for folate antagonists, including the anticancer drug methotrexate. In order to design novel drugs with altered binding properties, a detailed description of protein-drug interactions in solution is desirable to understand the specificity of drug binding. As a first step in this process, heteronuclear three-dimensional NMR spectroscopy has been used to make sequential resonance assignments for more than 90% of the residues in human dihydrofolate reductase complexed with methotrexate. Uniform enrichment of the 21.5-kDa protein with 15N was required to obtain the resonance assignments via heteronuclear 3D NMR spectroscopy since homonuclear 2D spectra did not provide sufficient resonance dispersion. Medium-and long-range NOE’s have been used to characterize the secondary structure of the binary ligand-enzyme complex in solution.Dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5. 1.3) catalyzes the NADPH-dependent reduction of 7, 8-dihydrofolate to 5, 6, 7, 8-tetrahydrofolate. Tetrahydrofolate or its derivatives are essential cofactors in the biosynthesis of purine nucleotides, thymidylate, and several amino acids. Failure to maintain adequate levels of tetra-