Gronenborn A, Birdsall B, Hyde E I, Roberts G C, Feeney J, Burgen A S
Biochemistry. 1981 Mar 31;20(7):1717-22. doi: 10.1021/bi00510a003.
The effects of coenzyme binding on the seven histidine C2 proton resonances of Lactobacillus casei dihydrofolate reductase have been determined. Binary complexes containing NADP+, NADPH, and their hypoxanthine, thionicotinamide, and acetylpyridine analogues, together with ternary complexes containing the inhibitors trimethoprim or methotrexate, have been examined. Four of the histidine residues are affected by coenzyme binding. The largest effect-a marked upfield shift (0.85 ppm) of the C2 proton resonance-is seen for His-64. The hypoxanthine analogue of the coenzyme was found to produce a smaller upfield shift and, in addition, a decrease in the pK of His-64. The effects on this reductase are discussed in the light of the crystal structure [Matthews, D. A., Alden, R. A., Bolin, J. T., Filman, D. J., Freer, S. T., Hamlin, R., Hol, W. G. J., Kisliuk, R. L., Pastore, E. J., Plante, L. T., Xuong, N., & Kraut, J. (1978) J. Biol. Chem. 253, 6946], and it is concluded that His-64 is close to a carboxyl group in the free enzyme and that the hypoxanthine ring binds in a somewhat different orientation to the adenine ring. The effects on histidine resonances A, E, and G are significantly different for oxidized and reduced coenzymes. The changes in pK of the histidines giving rise to resonances A and E (probably His-22 and His-18) are discussed in terms of ligand-induced conformational changes, which differ for NADP+ and NADPH.
已确定辅酶结合对干酪乳杆菌二氢叶酸还原酶七个组氨酸C2质子共振的影响。研究了含有NADP⁺、NADPH及其次黄嘌呤、硫代烟酰胺和乙酰吡啶类似物的二元复合物，以及含有抑制剂甲氧苄啶或甲氨蝶呤的三元复合物。四个组氨酸残基受辅酶结合的影响。辅酶结合对His-64的影响最大，其C2质子共振出现明显的高场位移（0.85 ppm）。发现辅酶的次黄嘌呤类似物产生较小的高场位移，此外，His-64的pK值降低。根据晶体结构[Matthews, D. A., Alden, R. A., Bolin, J. T., Filman, D. J., Freer, S. T., Hamlin, R., Hol, W. G. J., Kisliuk, R. L., Pastore, E. J., Plante, L. T., Xuong, N., & Kraut, J. (1978) J. Biol. Chem. 253, 6946]对这种还原酶的影响进行了讨论，得出的结论是，在游离酶中His-64靠近一个羧基，并且次黄嘌呤环与腺嘌呤环的结合方向略有不同。氧化型和还原型辅酶对组氨酸共振A、E和G的影响有显著差异。根据配体诱导的构象变化讨论了产生共振A和E的组氨酸（可能是His-22和His-18）pK值的变化，NADP⁺和NADPH的构象变化不同。
