T7 RNA聚合酶的C末端羧基可确保高效的镁离子依赖性催化作用。
The C-terminal carboxy group of T7 RNA polymerase ensures efficient magnesium ion-dependent catalysis.
作者信息
Lykke-Andersen J, Christiansen J
机构信息
RNA Regulation Centre, Institute of Molecular Biology, University of Copenhagen, Solvgade 83 H,DK-1307 Copenhagen K, Denmark.
出版信息
Nucleic Acids Res. 1998 Dec 15;26(24):5630-5. doi: 10.1093/nar/26.24.5630.
DNA and RNA polymerases use divalent metal ions for catalysis. Crystal structures of several polymerases reveal that two acidic residues are involved in coordinating two metal ions at the catalytic centre. Bacteriophage RNA polymerases contain a highly conserved C-terminus with the carboxylate positioned near the active site. We examined whether theC-terminal carboxy group of T7 RNA polymerase is important for magnesium ion-dependent catalysis. Introduction of a methyl ester or decarboxylation of the C-terminal carboxy group was achieved with an intein-based protein expression system and an elongation rate assay was developed to test the effects of the modifications. The results show that enzymes with a modified C-terminal carboxy group exhibit a magnesium ion-dependent decrease in catalytic activity.
DNA和RNA聚合酶利用二价金属离子进行催化。几种聚合酶的晶体结构表明，两个酸性残基参与在催化中心配位两个金属离子。噬菌体RNA聚合酶含有一个高度保守的C末端，其羧酸盐位于活性位点附近。我们研究了T7 RNA聚合酶的C末端羧基对于镁离子依赖性催化是否重要。利用基于内含肽的蛋白质表达系统实现了C末端羧基的甲酯化或脱羧，并开发了延伸率测定法来测试修饰的效果。结果表明，具有修饰的C末端羧基的酶在催化活性上呈现出镁离子依赖性降低。
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