Catalytic cycle of human glutathione reductase near 1 A resolution.
Catalytic cycle of human glutathione reductase near 1 A resolution.
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DOI：
10.1016/j.jmb.2008.06.083
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发表时间：
2008-10-03
影响因子：
5.6
通讯作者：
Karplus, P. Andrew
中科院分区：
文献类型：
--
作者：
Berkholz, Donald S.;Faber, H. Richard;Savvides, Savvas N.;Karplus, P. Andrew
有效的酶催化取决于结构的精致细节，超出了典型中高分辨率晶体分析所能解析的范围。在此，我们报告了基于同步加速器的冷冻晶体学研究，对黄素酶人谷胱甘肽还原酶 (GR) 的天然底物复合物进行了标称分辨率在 1.1 至 0.95 Å 之间的研究，揭示了其机制的新方面。活性位点的压缩会导致范德华半径重叠，并导致 NADPH 底物的烟酰胺环变形，从而通过立体电子效应增强催化作用。结合的 NADPH 和氧化还原活性二硫化物最佳地位于黄素的相对侧，以便在黄素双键上进行 1,2-加成。新结构扩展了早期的观察结果，揭示了GR中的氧化还原活性二硫键环是连续肽键系统性偏离平面性的极端情况，5个残基的净偏差为53°。但这种明显的应变并不是催化的一个因素，因为它存在于氧化和还原结构中。有趣的是，氧化GR中的黄素键长介于氧化黄素和还原黄素的预期值之间，但我们提供的证据表明，这可能不是由于蛋白质环境，而是由于同步加速器光束对黄素的部分同步还原。最后，具有更普遍的相关性，我们提出的证据表明，同步加速器还原的二硫键的结构通常不能用作自然还原的二硫键的可靠模型。
Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 Å that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity, a net deviation of 53° across 5 residues. But this apparent strain is not a factor in catalysis as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds.
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DOI：
10.1107/s0907444999016261
发表时间：
2000-03-01
影响因子：
2.2
作者：
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通讯作者：
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影响因子：
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作者：
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DOI：
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发表时间：
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影响因子：
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作者：
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通讯作者：
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影响因子：
56.9
作者：
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通讯作者：
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影响因子：
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作者：
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