Department of Chemistry, Georgia State University, Atlanta, Georgia 30302, United States.
Biochemistry. 2011 Aug 16;50(32):7033-44. doi: 10.1021/bi200456u. Epub 2011 Jul 21.
Post-translational modifications (PTMs) are important strategies used by eukaryotic organisms to modulate their phenotypes. One of the well-studied PTMs, arginine methylation, is catalyzed by protein arginine methyltransferases (PRMTs) with SAM as the methyl donor. The functions of PRMTs have been broadly studied in different biological processes and diseased states, but the molecular basis for arginine methylation is not well-defined. In this study, we report the transient-state kinetic analysis of PRMT1 catalysis. The fast association and dissociation rates suggest that PRMT1 catalysis of histone H4 methylation follows a rapid equilibrium sequential kinetic mechanism. The data give direct evidence that the chemistry of methyl transfer is the major rate-limiting step and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, from the stopped-flow fluorescence measurements, we have identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. These results provide new insights into the mechanism of arginine methylation and the rational design of PRMT inhibitors.
翻译后修饰（PTMs）是真核生物用于调节其表型的重要策略。精氨酸甲基化是其中一种经过充分研究的翻译后修饰，由蛋白质精氨酸甲基转移酶（PRMTs）以SAM作为甲基供体催化。PRMTs的功能已在不同生物过程和疾病状态中得到广泛研究，但精氨酸甲基化的分子基础尚未明确界定。在本研究中，我们报告了PRMT1催化的瞬态动力学分析。快速的结合和解离速率表明，PRMT1对组蛋白H4的甲基化催化遵循快速平衡顺序动力学机制。数据直接证明甲基转移的化学反应是主要的限速步骤，并且辅因子SAM或SAH的结合会影响H4与PRMT1的结合和解离。重要的是，通过停流荧光测量，我们确定了一个关键的动力学步骤，表明底物结合诱导了催化前的构象转变。这些结果为精氨酸甲基化机制及PRMT抑制剂的合理设计提供了新的见解。
