Abstract：
Small cationic guanyl-preferring ribonucleases (RNases) produced by the Bacillus species share a similar protein tertiary structure with a high degree of amino acid sequence conservation. However, they form dimers that differ in conformation and stability. Here, we have addressed the issues (1) whether the homologous RNases also have distinctions in catalytic activity towards different RNA substrates and interactions with the inhibitor protein barstar, and (2) whether these differences correlate with structural features of the proteins. Circular dichroism and dynamic light scattering assays revealed distinctions in the structures of homologous RNases. The activity levels of the RNases towards natural RNA substrates, as measured spectrometrically by acid-soluble hydrolysis products, were similar and decreased in the row high-polymeric RNA >>> transport RNA > double-stranded RNA. However, stopped flow kinetic studies on model RNA substrates containing the guanosine residue in a hairpin stem or a loop showed that the cleavage rates of these enzymes were different. Moreover, homologous RNases were inhibited by the barstar with diverse efficiency. Therefore, minor changes in structure elements of homologous proteins have a potential to significantly effect molecule stability and functional activities, such as catalysis or ligand binding.
摘要：
由芽孢杆菌属物种产生的小阳离子鸟苷酸偏好核糖核酸酶（RNase）具有相似的蛋白质三级结构，具有高度的氨基酸序列保守性。然而,它们形成构象和稳定性不同的二聚体。这里,我们已经解决了以下问题：（1）同源RNases对不同RNA底物的催化活性以及与抑制剂蛋白barstar的相互作用是否也有区别，（2）这些差异是否与蛋白质的结构特征相关。圆二色性和动态光散射测定揭示了同源RNase结构的区别。RNA酶对天然RNA底物的活性水平，通过酸溶性水解产物进行光谱测定，在高聚RNA>>>转运RNA>双链RNA的行中相似且减少。然而,对发夹茎或环中含有鸟苷残基的模型RNA底物的停止流动动力学研究表明，这些酶的裂解速率不同。此外，同源RNases被barstar抑制，效率不同。因此,同源蛋白质的结构元件的微小变化具有显著影响分子稳定性和功能活性的潜力，如催化或配体结合。
