Mechanistic studies on transcriptional coactivator protein arginine methyltransferase 1.
Mechanistic studies on transcriptional coactivator protein arginine methyltransferase 1.
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DOI：
10.1021/bi102022e
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发表时间：
2011-04-26
期刊：
影响因子：
2.9
通讯作者：
Thompson PR
中科院分区：
文献类型：
--
作者：
Rust HL;Zurita-Lopez CI;Clarke S;Thompson PR
蛋白质精氨酸甲基转移酶（PRMT）催化许多重要的细胞信号蛋白中的甲基从S-腺苷甲硫氨酸（SAM）转移到精氨酸残基的胍基。PRMT 1是该家族的创始成员，其活性在心脏病和癌症中似乎失调。为了开始表征这种同工酶的催化机制，我们评估了突变许多高度保守的活性位点残基（即，Y39、R54、E100、E144、E153、M155和H293），其被认为在SAM识别、底物结合和催化中起关键作用。这些研究的结果，以及pH速率的研究，和溶剂同位素效应（SIE）的测定，表明M155在SAM结合和反应的持续合成能力中起着关键作用，但不负责区域特异性形成的不对称二甲基精氨酸（ADMA）。此外，对H293的诱变研究，结合pH值研究和缺乏正常SIE，不支持该残基作为通用碱基的作用。此外，WT或催化受损突变体缺乏正常的SIE表明，一般的酸/碱催化对于促进甲基转移并不重要。该结果，结合E144 A/E153 A双突变体比单独的单突变体保留了显著更多的活性的事实，表明PRMT 1催化的反应主要是通过使底物胍盐与SAM的S-甲基紧密接近来驱动的，并且甲基转移不需要底物胍盐的预先去质子化。
Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to the guanidinium group of arginine residues in a number of important cell signaling proteins. PRMT1 is the founding member of this family and its activity appears to be dysregulated in heart disease and cancer. To begin to characterize the catalytic mechanism of this isozyme, we assessed the effects of mutating a number of highly conserved active site residues (i.e., Y39, R54, E100, E144, E153, M155, and H293), which are believed to play key roles in SAM recognition, substrate binding, and catalysis. The results of these studies, as well as pH rate studies, and the determination of solvent isotope effects (SIEs), indicate that M155 plays a critical role in both SAM binding and the processivity of the reaction, but is not responsible for the regiospecific formation of asymmetrically dimethylated arginine (ADMA). Additionally, mutagenesis studies on H293, combined with pH studies and the lack of a normal SIE, do not support a role for this residue as a general base. Furthermore, the lack of a normal SIE with either the WT or catalytically impaired mutants suggests that general acid/base catalysis is not important for promoting methyl transfer. This result, combined with the fact that the E144A/E153A double mutant retains considerably more activity then the single mutants alone, suggests that the PRMT1 catalyzed reaction is primarily driven by bringing the substrate guanidinium into close proximity to the S-methyl group of SAM and that the prior deprotonation of the substrate guanidinium is not required for methyl transfer.
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