The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition
The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition
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DOI：
10.1021/bi002438h
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发表时间：
2001-02-20
期刊：
影响因子：
2.9
通讯作者：
Eisenberg, D
中科院分区：
文献类型：
--
作者：
Gill, HS;Eisenberg, D
膦丝菌素是谷氨酰胺合成酶（GS）的有效抑制剂。将鼠伤寒沙门氏菌GS天然结构的分辨率提高到2.5埃，并将改进的模型用于差示傅立叶方法测定GS与膦丝菌素复合物的结构。该结构表明非共价的死端抑制机制。膦丝菌素占据谷氨酸底物口袋，并将Glu327瓣稳定在阻止谷氨酸进入活性位点的位置，将抑制剂捕获在酶上。膦丝菌素的氧膦基的一个氧似乎是质子化的，因为它接近Glu 327的羧酸基团。另一个氧膦基氧突出到带负电荷的底物铵结合口袋中，破坏该口袋。谷氨酸结合口袋中的电荷分布与膦丝菌素的电荷分布互补。活性位点内第二个铵结合位点的存在通过其类似物亚铊离子确认，标记铵位点及其蛋白质配体。甲硫氨酸亚砜亚胺对GS的抑制作用可以用同样的机制来解释。这些抑制GS的模型进一步阐明了其催化机制。
Phosphinothricin is a potent inhibitor of the enzyme glutamine synthetase (GS). The resolution of the native structure of GS from Salmonella typhimurium has been extended to 2.5 Angstrom resolution, and the improved model is used to determine the structure of phosphinothricin complexed to GS by difference Fourier methods. The structure suggests a noncovalent, dead-end mechanism of inhibition. Phosphinothricin occupies the glutamate substrate pocket and stabilizes the Glu327 flap in a position which blocks the glutamate entrance to the active site, trapping the inhibitor on the enzyme. One oxygen of the phosphinyl group of phosphinothricin appears to be protonated, because of its proximity to the carboxylate group of Glu327. The other phosphinyl oxygen protrudes into the negatively charged binding pocket for the substrate ammonium, disrupting that pocket. The distribution of charges in the glutamate binding pocket is complementary to those of phosphinothricin. The presence of a second ammonium binding site within the active site is confirmed by its analogue thallous ion, marking the ammonium site and its protein ligands. The inhibition of GS by methionine sulfoximine can be explained by the same mechanism. These models of inhibited GS further illuminate its catalytic mechanism.