Baeza Josue, Smallegan Michael J, Denu John M
Department of Biomolecular Chemistry and ‡Wisconsin Institute for Discovery, University of Wisconsin , Madison, Wisconsin 53715, United States.
ACS Chem Biol. 2015 Jan 16;10(1):122-8. doi: 10.1021/cb500848p.
Protein acetylation of lysine ε-amino groups is abundant in cells, particularly within mitochondria. The contribution of enzyme-catalyzed and nonenzymatic acetylation in mitochondria remains unresolved. Here, we utilize a newly developed approach to measure site-specific, nonenzymatic acetylation rates for 90 sites in eight native purified proteins. Lysine reactivity (as second-order rate constants) with acetyl-phosphate and acetyl-CoA ranged over 3 orders of magnitude, and higher chemical reactivity tracked with likelihood of dynamic modification in vivo, providing evidence that enzyme-catalyzed acylation might not be necessary to explain the prevalence of acetylation in mitochondria. Structural analysis revealed that many highly reactive sites exist within clusters of basic residues, whereas lysines that show low reactivity are engaged in strong attractive electrostatic interactions with acidic residues. Lysine clusters are predicted to be high-affinity substrates of mitochondrial deacetylase SIRT3 both in vitro and in vivo. Our analysis describing rate determination of lysine acetylation is directly applicable to investigate targeted and proteome-wide acetylation, whether or not the reaction is enzyme catalyzed.
赖氨酸ε-氨基的蛋白质乙酰化在细胞中很常见，尤其是在线粒体内。线粒体中酶催化乙酰化和非酶促乙酰化的作用仍未明确。在此，我们采用一种新开发的方法来测量8种天然纯化蛋白质中90个位点的位点特异性非酶促乙酰化速率。赖氨酸与乙酰磷酸和乙酰辅酶A的反应活性（作为二级速率常数）范围超过3个数量级，较高的化学反应活性与体内动态修饰的可能性相关，这表明酶催化酰化可能并非解释线粒体中乙酰化普遍存在所必需的。结构分析表明，许多高反应性位点存在于碱性残基簇中，而低反应性的赖氨酸则与酸性残基存在强烈的吸引性静电相互作用。赖氨酸簇在体外和体内均被预测为线粒体脱乙酰酶SIRT3的高亲和力底物。我们描述赖氨酸乙酰化速率测定的分析方法可直接用于研究靶向和全蛋白质组的乙酰化，无论该反应是否由酶催化。
