Wan Qun, Bennett Brad C, Wilson Mark A, Kovalevsky Andrey, Langan Paul, Howell Elizabeth E, Dealwis Chris
Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Disease, Yangzhou 225001, People's Republic of China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, People's Republic of China; Department of Biochemistry, College of Medicine, Yangzhou University, Yangzhou 225001, People's Republic of China;
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908;
Proc Natl Acad Sci U S A. 2014 Dec 23;111(51):18225-30. doi: 10.1073/pnas.1415856111. Epub 2014 Dec 1.
Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP(+). The neutron data were collected to 2.0-Å resolution using a 3.6-mm(3) crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed.
二氢叶酸还原酶（DHFR）催化二氢叶酸（DHF）依赖NADPH还原为四氢叶酸（THF）。该机制中的一个重要步骤涉及向DHF的N5原子提供质子。由于无法确定活性位点残基和底物的质子化状态，导致关于所涉及的催化机制缺乏共识。为了解决这一模糊性，我们对大肠杆菌DHFR与叶酸和NADP(+)的假米氏三元复合物进行了中子和超高分辨率X射线晶体学研究。使用3.6 mm³的晶体，采用准劳厄技术收集了分辨率为2.0 Å的中子数据。结构显示叶酸的N3原子被质子化，而Asp27带负电荷。先前的机制提出底物发生酮式到烯醇式互变异构以促进N5原子的质子化。该结构支持由于N3原子的质子化而存在酮式互变异构体，这表明互变异构对于催化不是必需的。在三元复合物分辨率为1.05 Å的X射线结构中，Met20侧链的构象无序与叶酸N5原子氢键距离内部分占据的水的电子密度相关联；这表明溶剂直接使底物质子化。我们提出了一种DHFR的催化机制，该机制涉及底物酮式互变异构体的稳定、Asp27提高DHF的N5原子的pKa值以及通过Met20侧链的波动进入活性位点的水使N5质子化，即使Met20环是闭合的。
