大肠杆菌二氢叶酸还原酶的晶体结构：NADP⁺全酶和叶酸·NADP⁺三元复合物。底物结合及过渡态模型。

Crystal structures of Escherichia coli dihydrofolate reductase: the NADP+ holoenzyme and the folate.NADP+ ternary complex. Substrate binding and a model for the transition state.

作者信息

Bystroff C, Oatley S J, Kraut J

机构信息

Department of Chemistry, University of California, San Diego, La Jolla 92093.

出版信息

Biochemistry. 1990 Apr 3;29(13):3263-77. doi: 10.1021/bi00465a018.

DOI:10.1021/bi00465a018

PMID:2185835

Abstract

The crystal structure of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli has been solved as the binary complex with NADP+ (the holoenzyme) and as the ternary complex with NADP+ and folate. The Bragg law resolutions of the structures are 2.4 and 2.5 A, respectively. The new crystal forms are nonisomorphous with each other and with the methotrexate binary complex reported earlier [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. In general, NADP+ and folate binding conform to predictions, but the nicotinamide moiety of NADP+ is disordered in the holoenzyme and ordered in the ternary complex. A mobile loop (residues 16-20) involved in binding the nicotinamide is also disordered in the holoenzyme. We report a detailed analysis of the binding interactions for both ligands, paying special attention to several apparently strained interactions that may favor the transition state for hydride transfer. Hypothetical models are presented for the binding of 7,8-dihydrofolate in the Michaelis complex and for the transition-state complex.

摘要

已解析出大肠杆菌二氢叶酸还原酶（EC 1.5.1.3）与NADP⁺形成的二元复合物（全酶）以及与NADP⁺和叶酸形成的三元复合物的晶体结构。这些结构的布拉格定律分辨率分别为2.4 Å和2.5 Å。新的晶体形式彼此之间以及与先前报道的甲氨蝶呤二元复合物[Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650 - 13662]均为非同晶型。一般来说，NADP⁺和叶酸的结合符合预测，但NADP⁺的烟酰胺部分在全酶中无序，而在三元复合物中有序。参与结合烟酰胺的一个可移动环（残基16 - 20）在全酶中也无序。我们报告了对两种配体结合相互作用的详细分析，特别关注了一些可能有利于氢化物转移过渡态的明显紧张相互作用。给出了米氏复合物中7,8 - 二氢叶酸结合以及过渡态复合物的假设模型。