Heaphy S, Singh M, Gait M J
Biochemistry. 1987 Mar 24;26(6):1688-96. doi: 10.1021/bi00380a030.
Preparation and analysis of a series of mutants of bacteriophage T4 RNA ligase that carry single amino acid changes at or near the site of covalent reaction with ATP (adenylylation) are described. The mutant proteins were constructed by site-directed mutagenesis of the gene for T4 RNA ligase (g63) cloned in M13 vectors, transfer of the mutant genes into a lambda pL-containing expression plasmid, and subsequent expression in Escherichia coli. The results give further evidence that Lys-99 is the adenylylation site and that the residue is also important to step 3 in the RNA ligase mechanism (ligation between acceptor and adenylylated donor). Mutations at Glu-100 or Asp-101 have no effect on adenylylation, but Asp-101 is shown to be crucial to both step 2 (transfer of adenylyl to donor) and step 3.
本文描述了一系列噬菌体T4 RNA连接酶突变体的制备与分析，这些突变体在与ATP（腺苷酸化）发生共价反应的位点或其附近携带单个氨基酸变化。通过对克隆于M13载体中的T4 RNA连接酶基因（g63）进行定点诱变、将突变基因转移至含λ pL的表达质粒中并随后在大肠杆菌中表达，构建了突变蛋白。结果进一步证明Lys-99是腺苷酸化位点，且该残基对RNA连接酶机制的第3步（受体与腺苷酸化供体之间的连接）也很重要。Glu-100或Asp-101处的突变对腺苷酸化没有影响，但Asp-101对第2步（腺苷酰基转移至供体）和第3步均至关重要。
