Trievel R C, Li F Y, Marmorstein R
Department of Biochemistry and Biophysics, Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
Anal Biochem. 2000 Dec 15;287(2):319-28. doi: 10.1006/abio.2000.4855.
Histone N-acetyltransferases (HATs) are a group of enzymes which acetylate specific lysine residues in the N-terminal tails of nucleosomal histones to promote transcriptional activation. Recent structural and enzymatic work on the GCN5/PCAF HAT family has elucidated the structure of their catalytic domain and mechanism of histone acetylation. However, the substrate specificity of these enzymes has not been quantitatively investigated. Utilizing a novel microplate fluorescent HAT assay which detects the enzymatic production of coenzyme A (CoA), we have compared the activities of the HAT domains of human PCAF and its GCN5 homologue from yeast and Tetrahymena and found that they have similar kinetic parameters. PCAF was further assayed with a series of different length histone H3 peptide substrates, which revealed that the determinants for substrate recognition lie within a 19-residue sequence. Finally, we evaluated the acetylation of three putative PCAF substrates, histones H3 and H4 and the transcription factor p53, and have determined that histone H3 is significantly preferred over the histone H4 and p53 substrates. Taken together, the fluorescent acetyltransferase assay presented here should be widely applicable to other HAT enzymes, and the results obtained with PCAF demonstrate a strong substrate preference for the N-terminal residues of histone H3.
组蛋白N - 乙酰转移酶（HATs）是一类将核小体组蛋白N端尾巴上特定赖氨酸残基乙酰化以促进转录激活的酶。最近关于GCN5/PCAF HAT家族的结构和酶学研究阐明了其催化结构域的结构和组蛋白乙酰化机制。然而，这些酶的底物特异性尚未得到定量研究。利用一种新型的微孔板荧光HAT检测法来检测辅酶A（CoA）的酶促生成，我们比较了人类PCAF及其来自酵母和四膜虫的GCN5同源物的HAT结构域的活性，发现它们具有相似的动力学参数。用一系列不同长度的组蛋白H3肽底物对PCAF进行进一步检测，结果表明底物识别的决定因素位于一个19个残基的序列内。最后，我们评估了三种假定的PCAF底物，组蛋白H3和H4以及转录因子p53的乙酰化情况，确定组蛋白H3比组蛋白H4和p53底物更受青睐。综上所述，本文介绍的荧光乙酰转移酶检测法应广泛适用于其他HAT酶，并且用PCAF获得的结果表明其对组蛋白H3的N端残基有很强的底物偏好性。
