P-31, N-15, AND C-13 NMR OF GLYPHOSATE - COMPARISON OF PH TITRATIONS TO THE HERBICIDAL DEAD-END COMPLEX WITH 5-ENOLPYRUVOYLSHIKIMATE-3-PHOSPHATE SYNTHASE
P-31, N-15, AND C-13 NMR OF GLYPHOSATE - COMPARISON OF PH TITRATIONS TO THE HERBICIDAL DEAD-END COMPLEX WITH 5-ENOLPYRUVOYLSHIKIMATE-3-PHOSPHATE SYNTHASE
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DOI：
10.1021/bi00435a035
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发表时间：
1989-05-02
期刊：
影响因子：
2.9
通讯作者：
SIKORSKI, JA
中科院分区：
文献类型：
--
作者：
CASTELLINO, S;LEO, GC;SIKORSKI, JA
草甘膦 [N-(膦酰甲基)甘氨酸] 与 5-烯醇丙酮酰莽草酸-3-磷酸合酶 (EPSPS) (EC 2.5.1.19) 和底物莽草酸 3-磷酸 (S3P) 的除草死端三元复合物 (EGlyphS3P) 已通过 31P、15N 和 13C NMR 进行了表征。 EPSPS 结合草甘膦的 NMR 谱显示三个核中每一个都有独特的化学位移 (δ)。通过 31P NMR，死端复合物中的草甘膦是与游离草甘膦低场 3.5 ppm 不同的物质。该信号由 pH 10.1 和 -0.8 溶液中的游离草甘膦单独建模，如 31P δ 所示。与 pH 依赖性。死端复合物中草甘膦的 13C 信号[对于 C-1 [13C (99%)]] 比游离草甘膦的信号低场移动 4 ppm。这与自 13C δ以来羧酸盐的质子化不一致。 vs pH 依赖性决定模型 pH 为 10.1。死端复合物中草磷酸盐 (99%) 的 15N 信号比任何游离两性离子物质低 5 ppm，比 pH 10.1 时的平均游离物质低 10 ppm。 15N δ。对于死端复合物中的草甘膦，要求胺完全质子化。因此，草甘膦的膦酰甲基和羧甲基部分必须具有与pH 10.1或-0.8溶液中游离草甘膦相似的取向。使用 MacroModel 通过力场计算对草甘膦各离子态的结构进行建模。发现模拟的草甘膦-3 和+1 净带电物质与0、-1 和-2 物质非常相似且明显不同，预计它们会形成分子内氢键。对 31P .delta 进行关联。和 C-P-O 键角，以及 13C 和 15N δ。假设值分别与 C-C-O 和 C-N-C 键角相关。 EPSPS二元复合物中S3P的低场31P化学位移扰动（Δ=0.54）与结合后S3P的3-磷酸盐的电离一致。 然而，前场.DELTA..delta。当草甘膦与二元复合物结合形成死端复合物时，1.94 的值就会发生。与 S3P 31P .delta 的比较。 vs pH 滴定曲线主要指定 ES3P 二元复合物中 3-磷酸盐的二价阴离子，而 EGlyphS3P 复合物指示 3-磷酸盐处的净质子化。后一种类型的化学位移扰动可以通过 O-P-O 键角的变化来解释 [Gorenstein, D. G. (1984) Phosphorus-31 NMR, 原理和应用 (Gorenstein, D. G. Ed.) 第 1-33 页，Academic Press, Orlando, FL]。
The herbicidal dead-end ternary complex (EGlyphS3P) of glyphosate [N-(phosphonomethyl)glycine] with 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) (EC 2.5.1.19) and the substrate shikimate 3-phosphate (S3P) has been characterized by 31P, 15N, and 13C NMR. The NMR spectra of EPSPS-bound glyphosate show unique chemical shifts (.delta.) for each of the three nuclei. By 31P NMR, glyphosate in the dead-end complex is a distinct species 3.5 ppm downfield from free glyphosate. This signal is modeled by free glyphosate alone in solutions of pH 10.1 and -0.8 as shown by the 31P .delta. vs pH dependence. The 13C signal [for the C-1 [13C (99%)]] of glyphosate in the dead-end complex is shifted 4 ppm downfield from that of free glyphosate. This is not consistent with protonation of the carboxylate since the 13C .delta. vs pH dependence dictates a model pH of 10.1. The 15N signal for glyphosphate (99%) in the dead-end complex is 5 ppm further downfield than that of any free zwitterionic species and 10 ppm downfield from that of the average free species at pH 10.1. The 15N .delta. for glyphosate in the dead-end complex requires that the amine be fully protonated. Hence, the phosphonomethyl and carboxymethyl moieties of glyphosate must have orientations similar to that of glyphosate free in solution at pHs 10.1 or -0.8. The structures of each ionic state of glyphosate are modeled with force field calculations by using MacroModel. The modeled glyphosate-3 and +1 net charged species were found to be very similar and distinctly different from the 0, -1, and -2 species, which are predicted to form intramolecular H-bonds. A correlation is made for the 31P .delta. and the C-P-O bond angle, and the 13C and 15N .delta. values are postulated to be related to C-C-O and C-N-C bond angles, respectively. The downfield 31P chemical shift perturbation for S3P (.DELTA..delta. = 0.54) in the EPSPS binary complex is consistent with ionization of the 3-phosphate of S3P upon binding. However, an upfield .DELTA..delta. of 1.94 occurs when glyphosate binds to the binary complex, forming the dead-end complex. Comparison with the S3P 31P .delta. vs pH titration curve specifies predominantly the dianion of the 3-phosphate in the ES3P binary complex, while the EGlyphS3P complex indicates net protonation at the 3-phosphate. Chemical shift perturbations of this latter type may be explained by changes in the O-P-O bond angle [Gorenstein, D. G. (1984) Phosphorus-31 NMR, Principles and Applications (Gorenstein, D. G. Ed.) pp 1-33, Academic Press, Orlando, FL].