Das M K, Vithayathil P J
Biochim Biophys Acta. 1978 Mar 28;533(1):43-50. doi: 10.1016/0005-2795(78)90545-7.
The initial structural alteration of RNAase A due to acid denaturation (0.5 N HCl, 30 degrees C) that accompanies deamidation (without altering enzymic activity) has been dectected by spectrophotometric titration, fluorescence and ORD/CD measurements. It is shown that acid treated RNAase A has an altered conformation at neutral pH, 25 degrees C. This is characterized by the increased accessibility of buried tyrosine residue(s) towards the solvent. The most altered conformation of RNAase A is found in the 10 h acid-treated derivative. This has about 1.5 additional exposed tyrosine residues and a lesser amount of secondary structure than RNAase A. All three methods (titration, fluorescence and CD) established that the structural transition of RNAase A is biphasic. The first phase occurs within 1 h and the resulting subtle conformational change is constant up to 7 h. Following this, after the release of 0.55 mol of ammonia, the major conformational change begins. The altered conformation of the acid-denatured RNAase A could be reversed completely to the native state through a conformational change induced by substrate analogs like 2'- or 3'-CMP. Thus the monodeamidated derivative isolated from the acid-denatured RNAase A by phosphate is very similar to RNAase A in over-all conformation. The results suggest the possibility of flexibility in the RNAase A molecule that does not affect its catalytic activity, as probed through the tyrosine residues.
通过分光光度滴定、荧光以及ORD/CD测量，已检测到由于酸变性（0.5N HCl，30℃）伴随脱酰胺作用（而不改变酶活性）引起的核糖核酸酶A的初始结构改变。结果表明，经酸处理的核糖核酸酶A在25℃中性pH条件下具有改变的构象。其特征在于埋藏的酪氨酸残基对溶剂的可及性增加。核糖核酸酶A构象改变最大的是在经10小时酸处理的衍生物中。它比核糖核酸酶A多约1.5个额外暴露的酪氨酸残基，且二级结构数量较少。所有三种方法（滴定、荧光和圆二色）均证实核糖核酸酶A的结构转变是双相的。第一阶段在1小时内发生，所产生的细微构象变化直至7小时保持不变。在此之后，在释放出0.55摩尔氨后，主要的构象变化开始。酸变性的核糖核酸酶A的改变构象可通过底物类似物如2'-或3'-CMP诱导的构象变化完全逆转至天然状态。因此，通过磷酸盐从酸变性的核糖核酸酶A中分离出的单脱酰胺衍生物在总体构象上与核糖核酸酶A非常相似。结果表明，核糖核酸酶A分子存在不影响其催化活性的灵活性可能性，这是通过酪氨酸残基探测到的。
