Kolbanovskaia E Iu, Okorokov A L, Panov K I, Poliakov K M, Hartley R W, Karpeĭskiĭ M Ia
National Institute of Health, Bethesda, Md. 20892.
Mol Biol (Mosk). 1994 May-Jun;28(3):602-9.
Barnase, an extracellular ribonuclease produced by Bacillus amyloliquefaciens, belongs to a family of small microbial ribonucleases with similar structure and properties. These enzymes hydrolyze phosphodiester bonds on the 3' side of guanosine nucleotides in RNA. The guanylic specificity of barnase is more pronounced in the hydrolysis of dinucleotides or cyclonucleotide phosphates as substrates than in the hydrolysis of RNA or polynucleotides. To have an insight into the molecular basis of this phenomenon, we mutated amino acid residue Ser-57 in the "base recognition loop" of RNase Ba. The mutant protein was expressed in Escherichia coli producing system and purified for the study of the kinetic properties in the cleavage polynucleotide reactions. It was shown that the mutation of amino acid residue Ser-57 for Ala in the "recognition loop" of RNase Ba does not significantly influence the kinetic parametres of hydrolysis of polynucleotide substrates.
芽孢杆菌核糖核酸酶（Barnase）是解淀粉芽孢杆菌产生的一种细胞外核糖核酸酶，属于结构和性质相似的小型微生物核糖核酸酶家族。这些酶水解RNA中鸟苷酸3'侧的磷酸二酯键。与以RNA或多核苷酸为底物的水解反应相比，芽孢杆菌核糖核酸酶对鸟苷酸的特异性在以二核苷酸或环核苷酸磷酸为底物的水解反应中更为明显。为深入了解这一现象的分子基础，我们对核糖核酸酶Ba“碱基识别环”中的丝氨酸-57氨基酸残基进行了突变。突变蛋白在大肠杆菌表达系统中表达并纯化，用于研究其在切割多核苷酸反应中的动力学性质。结果表明，核糖核酸酶Ba“识别环”中丝氨酸-57氨基酸残基突变为丙氨酸对多核苷酸底物水解的动力学参数没有显著影响。
