Methotrexate and folate binding to dihydrofolate reductase. Separate characterization of the pteridine and p-aminobenzoyl binding sites by resonance Raman spectroscopy.
Methotrexate and folate binding to dihydrofolate reductase. Separate characterization of the pteridine and p-aminobenzoyl binding sites by resonance Raman spectroscopy.
复制标题
甲氨蝶呤和叶酸与二氢叶酸还原酶结合。
作者：
Y. Ozaki;R. King;P. Carey
通过使用324-和350.6-nm激发，可以分别在甲氨蝶呤（MTX）或叶酸内选择性地获得对氨基苯甲酰基和蝶啶发色团的共振拉曼光谱。因此，对于单个配体，通过改变激发波长，可以分别监测两个发色团的几何构象。共振拉曼光谱报告MTX结合二氢叶酸还原酶从大肠杆菌和干酪乳杆菌，在每种情况下，在存在和不存在的NADPH。此外，还提供了酶结合叶酸的一些数据。共振拉曼数据支持了其他工作者的结论，即MTX与质子化的蝶啶环结合，而叶酸内的蝶啶环作为中性物质结合。然而，对于MTX，显着的差异存在于质子化蝶啶环的配体在溶液中的自由和结合物种的电子分布。蝶啶电子的重排超过并高于伴随质子化解释了结合MTX的吸收性质，并且与质子化一起可以部分地解释MTX对酶的高亲和力。共振拉曼光谱表明，MTX在三种二氢叶酸还原酶中的蝶啶位点之间存在细微差异，而对氨基苯甲酰基位点之间没有差异。然而，在每个MTX-蛋白质复合物中，与游离配体中发现的几何形状相比，MTX的苯甲酰基连接中的酰胺基团的几何形状似乎发生了显著变化。甲氨蝶呤结合酶的共振拉曼光谱不变后，添加的辅因子NADPH，表明辅因子不带来显着的电子重排的结合配体。
By using 324- and 350.6-nm excitation, it is possible to obtain selectively the resonance Raman spectra of the p-aminobenzoyl and pteridine chromophores, respectively, within methotrexate (MTX) or folate. Thus, for a single ligand, by changing the wavelength for excitation, the geometric conformations of both chromophores can be monitored separately. Resonance Raman spectra are reported for MTX bound to dihydrofolate reductases from Escherichia coli and from Lactobacillus casei, in each case in the presence and absence of NADPH. Additionally, some data are presented for enzyme-bound folate. The resonance Raman data support the conclusions of other workers that MTX binds with its pteridine ring protonated while the pteridine ring within folate is bound as a neutral species. However, for MTX, marked differences exist between the electronic distribution in the protonated pteridine ring for the ligand free in solution and for the bound species. The rearrangement of the pteridine electrons over and above that accompanying protonation explains the absorption properties of bound MTX, and together with protonation may account in part for the high affinity of MTX for the enzyme. The resonance Raman spectra show that slight differences exists between the pteridine sites for MTX in the three dihydrofolate reductases studied while no differences could be detected among the p-aminobenzoyl sites. In each MTX-protein complex, however, there appears to be a marked change in the geometry of the amide group in the benzoyl linkage of MTX compared to the geometry found in the free ligand. The resonance Raman spectra of MTX bound to the enzymes were unchanged upon the addition of the cofactor NADPH, indicating that the cofactor does not bring about marked electron rearrangement in the bound ligands.