Kowieski Terri M, Lee Susan, Denu John M
Department of Biomolecular Chemistry, University of Wisconsin, School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
J Biol Chem. 2008 Feb 29;283(9):5317-26. doi: 10.1074/jbc.M707613200. Epub 2007 Dec 27.
Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.
沉默调节蛋白是一类高度保守的蛋白质家族，参与多种细胞过程，如基因沉默、衰老和代谢调节。尽管许多沉默调节蛋白催化一种特征明确的蛋白质/组蛋白去乙酰化反应，但有许多报道表明其具有蛋白质ADP-核糖基转移酶活性。在这里，我们以布氏锥虫Sir2同源物TbSIR2rp1作为据报道具有两种活性的沉默调节蛋白模型，探索了ADP-核糖基化的机制。稳态动力学分析显示其具有高活性的组蛋白去乙酰化酶（kcat = 0.1 s(-1)，NAD+的Km值为42微摩尔，乙酰化底物的Km值为65微摩尔）。一系列生化分析表明，TbSIR2rp1对蛋白质/组蛋白的ADP-核糖基化需要乙酰化底物。数据与两条不同的ADP-核糖基化途径一致，这两条途径涉及乙酰化底物、NAD+和TbSIR2rp1，具体如下：1）去乙酰化产物O-乙酰-ADP-核糖（或其水解产物ADP-核糖）与组蛋白之间的非催化反应，以及2）一种更有效的机制，涉及结合组蛋白的侧链亲核试剂拦截ADP-核糖-乙酰肽-酶中间体。然而，在相同条件下，两种ADP-核糖基化反应的总和比组蛋白去乙酰化慢约5个数量级。我们讨论了这些结果的生物学意义。
