Yakovlev G I, Karpeisky M Y, Bezborodova S I, Beletskaja O P, Sakharovsky V G
Eur J Biochem. 1980 Aug;109(1):75-85. doi: 10.1111/j.1432-1033.1980.tb04769.x.
The method of proton magnetic resonance was used to obtain information on the active site of the guanyl-specific ribonuclease from Penicillium chrysogenum, strain 152A. Four pH-dependent signals in the aromatic region of the proton NMR spectrum of the enzyme were assigned to the C-2 and C-4 protons of the two histidine residues. To determine the pK values and the environment of the histidine residues the pH dependence of their chemical shifts was studied and experimental curves thus obtained were analyzed taking into account the effect of other dissociating groups of the enzyme. The pK values of the histidine residues were found to be equal to 7.92 +/- 0.04 and 7.86 +/- 0.09. The results of the calculations indicate that each histidine residue should interact with an acidic group (carboxylic) of the protein (pK 4.33 and 3.48) and the distance between two histidine residues does not exceed 0.85 nm. The rate constants for the quasi-first order reaction of deuterium exchange of the histidine residues (11.2 s-1 and 3.7 x-1) suggest that both residues are accessible, though to a different degree to solvent. Formation of a complex between the enzyme and guanosine 3'-phosphate (Guo3'P) is accompanied by the shift of the histidine pK toward the alkaline region by 0.5. The existence of the complex is controlled by dissociation of a histidine residue with pK 8.7 in alkaline medium and by protonation of the N-7 of Guo3'P (pK 2.4) in acid medium. Nuclear Overhauser effect measurements were used to determine the glycosidic torsion angle for the Guo3'P in the complex and to estimate the distances between the histidine residues of the enzyme and ribose ring of Guo-3'P. The results obtained suggest that the nucleotide in the complex has an anti conformation and the least exposed histidine is spaced not more than 0.5 nm from the C-1' proton of the nucleotide ribose ring. A model for the enzyme-nucleotide complex is presented.
采用质子磁共振方法获取产黄青霉152A菌株的鸟苷特异性核糖核酸酶活性位点的信息。该酶质子核磁共振谱芳香区的四个pH依赖性信号被指定为两个组氨酸残基的C-2和C-4质子。为了确定组氨酸残基的pK值及其环境，研究了它们化学位移的pH依赖性，并在考虑酶的其他解离基团影响的情况下对由此获得的实验曲线进行了分析。发现组氨酸残基的pK值分别为7.92±0.04和7.86±0.09。计算结果表明，每个组氨酸残基应与蛋白质的酸性基团（羧基）相互作用（pK分别为4.33和3.48），且两个组氨酸残基之间的距离不超过0.85纳米。组氨酸残基氘交换的准一级反应速率常数（分别为11.2 s-1和3.7 s-1）表明，两个残基都可与溶剂接触，不过程度不同。酶与鸟苷3'-磷酸（Guo3'P）形成复合物时，组氨酸的pK向碱性区域移动0.5。复合物的存在受碱性介质中pK为8.7的组氨酸残基解离以及酸性介质中Guo3'P的N-7质子化（pK为2.4）的控制。利用核Overhauser效应测量来确定复合物中Guo3'P的糖苷扭转角，并估计酶的组氨酸残基与Guo-3'P核糖环之间的距离。所得结果表明，复合物中的核苷酸具有反式构象，且暴露程度最小的组氨酸与核苷酸核糖环的C-1'质子的间距不超过0.5纳米。本文提出了酶-核苷酸复合物的模型。
