Catalytic mechanism and function of invariant glutamic acid 173 from the histone acetyltransferase GCN5 transcriptional coactivator
Catalytic mechanism and function of invariant glutamic acid 173 from the histone acetyltransferase GCN5 transcriptional coactivator
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DOI：
10.1074/jbc.274.26.18157
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发表时间：
1999-06-25
影响因子：
4.8
通讯作者：
Denu, JM
中科院分区：
文献类型：
--
作者：
Tanner, KG;Trievel, RC;Denu, JM
在染色质内，核心组蛋白的可逆乙酰化对于真核靶基因的转录激活是至关重要的。最近从许多转录共激活因子（包括酵母GCN 5、p300/CBP、P/CAF和TAFII 250）中鉴定出内在组蛋白乙酰转移酶（HAT）催化活性，强调了蛋白乙酰化在转录控制中的重要性。GCN 5家族是至少四个不同人类HAT家族的多样化组的原型。虽然现在体内HAT催化活性和基因激活之间有明确的联系，但对组蛋白乙酰化的分子机制知之甚少。在此，我们报告了第一个详细的生化研究，探讨催化机制和功能不变的谷氨酸173在GCNB家族的HAT。我们的结果表明，HAT反应涉及形成一个三元复合物（组蛋白、乙酰辅酶A和酶），其中组蛋白赖氨酸残基的E-氨基直接攻击结合的乙酰辅酶A，乙酰化反应需要在亲核攻击之前使E-氨基去质子化。采用定点诱变，化学修饰，稳态，和pH依赖性速率分析，它表明，谷氨酸173是一个必不可少的催化残基，作为一个通用的碱催化剂通过去质子化的组蛋白底物。
Within chromatin, reversible acetylation of core histones is critical for transcriptional activation of eukaryotic target genes. The recent identification of intrinsic histone acetyltransferase (HAT) catalytic activity from a number of transcriptional co-activators (including yeast GCN5, p300/CBP, P/CAF, and TAFII250), has underscored the importance of protein acetylation in transcriptional control. The GCN5 family is the prototype for a diverse group of at least four distinct human HATs families. Although there is now a clear link between in vivo HAT catalytic activity and gene activation, little is known about the molecular mechanisms of histone acetylation. Herein, we report the first detailed biochemical study that probes the catalytic mechanism and the function of invariant glutamic acid 173 within the GCNB family of HATs, Our results suggest that the HAT reaction involves the formation of a ternary complex (histones, acetyl-CoA and enzyme) where the E-amino group of histone lysine residues directly attacks the bound acetyl-CoA, The acetylation reaction requires deprotonation of the E-amino group prior to nucleophilic attack. Employing site-directed mutagenesis, chemical modification, steady-state, and pH-dependent rate analysis, it is demonstrated that glutamic acid 173 is an essential catalytic residue, acting as a general base catalyst by deprotonating the histone substrate.