利用定点突变分析粘质沙雷氏菌核酸酶的作用机制。
Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis.
作者信息
Friedhoff P, Kolmes B, Gimadutdinow O, Wende W, Krause K L, Pingoud A
机构信息
Institut für Biochemie, Justus-Liebig-Universität, Giessen, Germany.
出版信息
Nucleic Acids Res. 1996 Jul 15;24(14):2632-9. doi: 10.1093/nar/24.14.2632.
Abstract
Based on crystal structure analysis of the Serratia nuclease and a sequence alignment of six related nucleases, conserved 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 kcat, others in their Km. 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.
摘要
基于粘质沙雷氏菌核酸酶的晶体结构分析以及六种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附近的保守氨基酸残基进行诱变研究。分析的12个氨基酸残基中有5个对该酶的催化活性尤为重要：Arg57、Arg87、His89、Asn119和Glu127。例如，用丙氨酸取代它们会导致活性极低的突变蛋白，其活性不到野生型酶活性的1%。对突变蛋白的稳态动力学分析表明，其中一些突变体主要受kcat影响，另一些则受Km影响。这些结果以及所选突变蛋白的pH和金属离子依赖性测定结果被用于初步确定这些氨基酸残基在粘质沙雷氏菌核酸酶切割磷酸二酯键机制中的功能。
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