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首页> 外文期刊>Nucleic Acids Research >ANALYSIS OF THE MECHANISM OF THE SERRATIA NUCLEASE USING SITE-DIRECTED MUTAGENESIS
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ANALYSIS OF THE MECHANISM OF THE SERRATIA NUCLEASE USING SITE-DIRECTED MUTAGENESIS

机译：利用位点定向诱变分析血清型核糖核酸酶的机制

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摘要

Based on crystal structure analysis of the Serratia nuclease and a sequence alignment of six related nucleases, consented amino acid residues that are located in proximity to the previously identified catalytic site residue His89 were selected for a mutagenesis study. Five out of 12 amino acid residues analyzed turned out to be of particular importance for the catalytic activity of the enzyme: Arg57, Arg87, His89, Asn119 and Glu127. Their replacement by alanine, for example, resulted in mutant proteins of very low activity, <1% of the activity of the wild-type enzyme. Steady-state kinetic analysis of the mutant proteins demonstrates that some of these mutants are predominantly affected in their k(cat), others in their K-m. These results and the determination of the pH and metal ion dependence of selected mutant proteins were used for a tentative assignment for the function of these amino acid residues in the mechanism of phosphodiester bond cleavage by the Serratia nuclease.
机译：基于沙雷氏菌核酸酶的晶体结构分析和六个相关核酸酶的序列比对，选择位于先前鉴定的催化位点残基His89附近的允许的氨基酸残基进行诱变研究。事实证明，分析的12个氨基酸残基中有5个对酶的催化活性特别重要：Arg57，Arg87，His89，Asn119和Glu127。例如，它们被丙氨酸替代，导致突变蛋白的活性非常低，不到野生型酶活性的1％。突变体蛋白的稳态动力学分析表明，这些突变体中的一些主要受其k（cat）影响，而其他一些受其k-m影响。这些结果以及对选定突变蛋白的pH和金属离子依赖性的测定被用于初步确定这些氨基酸残基在沙雷氏菌核酸酶切割磷酸二酯键的机理中的功能。

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