Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity.
Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity.
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DOI：
10.1074/jbc.ra120.015103
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发表时间：
2021-01
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影响因子：
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通讯作者：
Carrodeguas JA
中科院分区：
文献类型：
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作者：
Latorre-Muro P;Baeza J;Hurtado-Guerrero R;Hicks T;Delso I;Hernández-Ruiz C;Velázquez-Campoy A;Lawton AJ;Angulo J;Denu JM;Carrodeguas JA
已知乙酰化通过促进酶的逆反应（将磷酸烯醇式丙酮酸转化为草酰乙酸）来调节胞质磷酸烯醇式丙酮酸羧激酶（PCK1）的活性，所述磷酸烯醇式丙酮酸羧激酶（PCK1）是胚胎发生中的关键酶。也已知组蛋白乙酰转移酶p300可以诱导细胞中的PCK1乙酰化，但这是否是直接或间接的功能尚不清楚。在这里，我们首先确定p300是否可以在体外直接乙酰化PCK1。我们报告说，p300弱乙酰化PCK1，但令人惊讶的是，使用几种技术，包括蛋白质结晶，质谱，等温滴定量热法，饱和转移差核磁共振和分子对接，我们发现，PCK1也能够乙酰化本身使用乙酰辅酶A独立的p300。该反应产生了乙酰化的重组PCK1，其kcat降低3倍，而所有底物的Km均无变化。乙酰化化学计量测定的14个残基，包括残基内衬的活性位点。结构和动力学分析确定，K244的定点乙酰化，位于活性位点内，改变了这个网站，使酶失活。此外，我们发现乙酰辅酶A与活性位点的结合是特异性的和金属依赖性的。我们的研究结果提供了直接的证据乙酰辅酶A结合和化学反应的活性位点的PCK1，并提出了一个新发现的调节机制的PCK1在代谢应激。
Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.