Borah B, Chen C W, Egan W, Miller M, Wlodawer A, Cohen J S
Biochemistry. 1985 Apr 9;24(8):2058-67. doi: 10.1021/bi00329a038.
The complex of ribonuclease A (RNase A) with uridine vanadate (U-V), a transition-state analogue, has been studied with 51V and proton NMR spectroscopy in solution and by neutron diffraction in the crystalline state. Upon the addition of aliquots of U-V at pH 6.6, the C epsilon-H resonances of the two active-site histidine residues 119 and 12 decrease in intensity while four new resonances appear. Above pH 8 and below pH 5, these four resonances decrease in intensity as the complex dissociates. These four resonances are assigned to His-119 and His-12 in protonated and unprotonated forms in the RNase-U-V complex. These resonances do not titrate or change in relative area in the pH range 5-8, indicating a slow protonation process, and the extent of protonation remains constant with ca. 58% of His-12 and ca. 26% of His-119 being protonated. The results of diffraction studies show that both His-12 and His-119 occupy well-defined positions in the RNase-U-V complex and that both are protonated. However, while the classic interpretation of the mechanism of action of RNase based on the proposal of Findlay et al. [Findlay, D., Herries, D. G., Mathias, A. P., Rabin, B. R., & Ross, C. A. (1962) Biochem. J. 85, 152-153] requires both His-12 and His-119 to be in axial positions relative to the pentacoordinate transition state, in the diffraction structure His-12 is found to be in an equatorial position, while Lys-41 is close to an axial position. Hydrogen exchange data show that the mobility and accessibility of amides in the RNase-U-V complex do not significantly differ from what was observed in the native enzyme. The results of both proton NMR in solution and neutron diffraction in the crystal are compared and interpreted in terms of the mechanism of action of RNase.
已通过溶液中的(^{51}V)和质子核磁共振光谱以及晶体状态下的中子衍射对核糖核酸酶A（RNase A）与过渡态类似物钒酸尿苷（U-V）的复合物进行了研究。在pH 6.6下逐份添加U-V时，两个活性位点组氨酸残基119和12的(C\epsilon-H)共振强度降低，同时出现四个新的共振峰。在pH 8以上和pH 5以下，随着复合物解离，这四个共振峰的强度降低。这四个共振峰被归属于RNase-U-V复合物中质子化和未质子化形式的His-119和His-12。这些共振峰在pH 5-8范围内不发生滴定或相对面积变化，表明质子化过程缓慢，且质子化程度保持恒定，约58%的His-12和约26%的His-119被质子化。衍射研究结果表明，His-12和His-119在RNase-U-V复合物中均占据明确的位置，且二者均被质子化。然而，虽然基于Findlay等人的提议[Findlay, D., Herries, D. G., Mathias, A. P., Rabin, B. R., & Ross, C. A. (1962) Biochem. J. 85, 152 - 153]对RNase作用机制的经典解释要求His-12和His-119相对于五配位过渡态均处于轴向位置，但在衍射结构中发现His-12处于赤道位置，而Lys-41接近轴向位置。氢交换数据表明，RNase-U-V复合物中酰胺的流动性和可及性与天然酶中观察到的情况没有显著差异。对溶液中的质子核磁共振和晶体中的中子衍射结果进行了比较，并根据RNase的作用机制进行了解释。
