Hou Ya-Ming
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, BLSB 220, Philadelphia, PA, 19107-5719, USA.
Methods Mol Biol. 2016;1421:79-96. doi: 10.1007/978-1-4939-3591-8_8.
Methyl transfer from S-adenosyl methionine (abbreviated as AdoMet) to biologically active molecules such as mRNAs and tRNAs is one of the most fundamental and widespread reactions in nature, occurring in all three domains of life. The measurement of kinetic constants of AdoMet-dependent methyl transfer is therefore important for understanding the reaction mechanism in the context of biology. When kinetic constants of methyl transfer are measured in steady state over multiple rounds of turnover, the meaning of these constants is difficult to define and is often limited by non-chemical steps of the reaction, such as product release after each turnover. Here, the measurement of kinetic constants of methyl transfer by tRNA methyltransferases in rapid equilibrium binding condition for one methyl transfer is described. The advantage of such a measurement is that the meaning of kinetic constants can be directly assigned to the steps associated with the chemistry of methyl transfer, including the substrate binding affinity to the methyltransferase, the pre-chemistry re-arrangement of the active site, and the chemical step of methyl transfer. An additional advantage is that kinetic constants measured for one methyl transfer can be correlated with structural information of the methyltransferase to gain direct insight into its reaction mechanism.
从S-腺苷甲硫氨酸（简称为AdoMet）向诸如mRNA和tRNA等生物活性分子的甲基转移是自然界中最基本且广泛存在的反应之一，发生在生命的所有三个域中。因此，测定依赖AdoMet的甲基转移的动力学常数对于在生物学背景下理解反应机制很重要。当在多轮周转的稳态下测量甲基转移的动力学常数时，这些常数的含义难以界定，并且常常受到反应的非化学步骤的限制，例如每次周转后的产物释放。在此，描述了在快速平衡结合条件下针对一次甲基转移由tRNA甲基转移酶测量甲基转移的动力学常数。这种测量的优点在于，动力学常数的含义可以直接归因于与甲基转移化学相关的步骤，包括底物与甲基转移酶的结合亲和力、活性位点的化学前重排以及甲基转移的化学步骤。另一个优点是，针对一次甲基转移测量的动力学常数可以与甲基转移酶的结构信息相关联，以直接深入了解其反应机制。
