Sakaguchi Reiko, Lahoud Georges, Christian Thomas, Gamper Howard, Hou Ya-Ming
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10(th) Street, BLSB 220, Philadelphia, PA 19107, USA.
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10(th) Street, BLSB 220, Philadelphia, PA 19107, USA.
Chem Biol. 2014 Oct 23;21(10):1351-1360. doi: 10.1016/j.chembiol.2014.07.023. Epub 2014 Sep 11.
The catalytic mechanism of the majority of S-adenosyl methionine (AdoMet)-dependent methyl transferases requires no divalent metal ions. Here we report that methyl transfer from AdoMet to N(1) of G37-tRNA, catalyzed by the bacterial TrmD enzyme, is strongly dependent on divalent metal ions and that Mg(2+) is the most physiologically relevant. Kinetic isotope analysis, metal rescue, and spectroscopic measurements indicate that Mg(2+) is not involved in substrate binding, but in promoting methyl transfer. On the basis of the pH-activity profile indicating one proton transfer during the TrmD reaction, we propose a catalytic mechanism in which the role of Mg(2+) is to help to increase the nucleophilicity of N(1) of G37 and stabilize the negative developing charge on O(6) during attack on the methyl sulfonium of AdoMet. This work demonstrates how Mg(2+) contributes to the catalysis of AdoMet-dependent methyl transfer in one of the most crucial posttranscriptional modifications to tRNA.
大多数依赖S-腺苷甲硫氨酸（AdoMet）的甲基转移酶的催化机制不需要二价金属离子。在此我们报告，细菌TrmD酶催化的从AdoMet到G37-tRNA的N(1)的甲基转移强烈依赖于二价金属离子，且Mg(2+)是最具生理相关性的。动力学同位素分析、金属挽救和光谱测量表明，Mg(2+)不参与底物结合，而是促进甲基转移。基于pH-活性曲线表明TrmD反应过程中有一个质子转移，我们提出了一种催化机制，其中Mg(2+)的作用是帮助增加G37的N(1)的亲核性，并在攻击AdoMet的甲硫鎓时稳定O(6)上正在形成的负电荷。这项工作展示了Mg(2+)如何在tRNA最关键的转录后修饰之一中促进依赖AdoMet的甲基转移的催化作用。
