Dudkin S M, Karabachyan L V, Borisova S N, Shlyapnikov S V, Karpeisky M Y, Geidarov T G
Biochim Biophys Acta. 1975 Mar 28;386(1):275-82. doi: 10.1016/0005-2795(75)90269-x.
We have studied the spectral properties of RNAase A containing a phosphopyridoxyl residue at the epsilon-NH2 group of Lys-7 or Lys-14. The overall conformations of the native and modified enzymes were shown to be rather similar. All three proteins have similar circular dichroism spectra within the 220-300-nm region, and similar thermal transition temperatures. All the changes in the RNAase A molecule modified are located in close proximity to the alkylated lysine residue. The phosphopyridoxyl group of (P-Pxy)-epsilon-Lys-41-RNAase A is situated directly at the enzyme active site and is 25% butied in the protein globule. The P-pyridoxyl group of (P-Pxy)-epsilon-Lys-7-RNAase A was shown to be located in the vicinity of the active site and to be more exposed to the solvent. In the pyridoxyl phosphate absorption band, optical activity is induced in both proteins. Study of the pH dependence of the changes occurring in the circular dichroism and absorption spectra has shown that in the modified proteins, the pyridoxyl phosphate chromophore is rather sensitive to the ionic state of the surrounding medium and serves as a "reporter" group when the relationship between structure and function of the RNAase A active site is being investigated.
我们研究了在赖氨酸-7或赖氨酸-14的ε-NH₂基团上含有磷酸吡哆醛残基的核糖核酸酶A的光谱特性。结果表明，天然酶和修饰酶的整体构象相当相似。所有这三种蛋白质在220 - 300纳米区域内具有相似的圆二色光谱和相似的热转变温度。核糖核酸酶A分子中修饰的所有变化都位于烷基化赖氨酸残基附近。（P-Pxy）-ε-赖氨酸-41-核糖核酸酶A的磷酸吡哆醛基团直接位于酶的活性位点，在蛋白质球状体中被埋藏了25%。（P-Pxy）-ε-赖氨酸-7-核糖核酸酶A的磷酸吡哆醛基团被证明位于活性位点附近，且更暴露于溶剂中。在磷酸吡哆醛吸收带中，两种蛋白质都诱导出了光学活性。对圆二色光谱和吸收光谱中发生的变化的pH依赖性研究表明，在修饰的蛋白质中，磷酸吡哆醛发色团对周围介质的离子状态相当敏感，并且在研究核糖核酸酶A活性位点的结构与功能之间的关系时可作为一个“报告”基团。
