Quantitating the specificity and selectivity of Gcn5-mediated acetylation of histone H3.
Quantitating the specificity and selectivity of Gcn5-mediated acetylation of histone H3.
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DOI：
10.1371/journal.pone.0054896
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Andrews AJ
中科院分区：
文献类型：
--
作者：
Kuo YM;Andrews AJ
赖氨酸乙酰基转移酶（KATs）在调控基因转录和维持细胞表观遗传状态方面具有独特的作用，如Gcn 5和p300/CBP可以修饰单个组蛋白上的多个残基;然而，乙酰化的顺序和特异性可以被诸如组蛋白伴侣、亚基蛋白或外部刺激等因素改变。难以定量测量组蛋白内不同残基处乙酰化的特异性和选择性。在本文中，我们展示了一种无标记的定量高通量质谱为基础的分析能够定量监测所有已知的乙酰化位点的H3同时。使用该测定，我们能够分析Gcn 5的稳态酶动力学，Gcn 5是一种进化上保守的KAT。在此过程中，我们测量了Gcn 5介导的六个残基的乙酰化（K14>K9> K23> K18> K27> K36）和K9、K14、K18和K23的催化效率（kcat/Km）以及非酶乙酰化速率。我们观察到K14和K18之间的选择性差异高达−4 kcal/mol，这是可测量的最高和最低kcat/Km。这些数据提供了通过Gcn 5对单一底物（H3）上的多个赖氨酸的特异性和选择性进行定量的第一视角。
Lysine acetyltransferases (KATs) play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14>K9 ≈ K23> K18> K27 ≈ K36) and the catalytic efficiency (kcat/Km) for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to −4 kcal/mol between K14 and K18, the highest and lowest measurable kcat/Km. These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3) by Gcn5.
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