The Krebs cycle enzyme fumarase is a dual-targeted protein that is located in the mitochondria and cytoplasm of eukaryotic cells. Besides being involved in the TCA cycle and primary metabolism, fumarase is a tumour suppressor that aids DNA repair in human cells. Using mass spectrometry, we identified modifications in peptides of cytosolic yeast fumarase, some of which were absent when the cells were exposed to DNA damage (using the homing endonuclease system or hydroxyurea). We show that DNA damage increased the enzymatic activity of fumarase, which we hypothesized to be affected by post-translational modifications. Succinylation and ubiquitination of fumarase at lysines 78 and 79, phosphorylation at threonine 122, serine 124 and threonine 126 as well as deamidation at arginine 239 were found to be functionally relevant. Upon homology analysis, these residues were also found to be evolutionally conserved. Serine 128, on the other hand, is not evolutionary conserved and the Fum1S128D phosphorylation mimic was able to aid DNA repair. Our molecular model is that the above modifications inhibit the enzymatic activity of cytosolic fumarase under conditions of no DNA damage induction and when there is less need for the enzyme. Upon genotoxic stress, some fumarase modifications are removed and some enzymes are degraded while unmodified proteins are synthesized. This report is the first to demonstrate how post-translational modifications influence the catalytic and DNA repair functions of fumarase in the cell.

克雷布斯循环酶富马酸酶是一种双重靶向蛋白，位于真核细胞的线粒体和细胞质中。除了参与TCA周期和初级代谢，富马酸酶还是一种肿瘤抑制因子，有助于人类细胞中的DNA修复。使用质谱法，我们确定了胞质酵母富马酸酶肽的修饰，当细胞暴露于DNA损伤（使用归巢内切核酸酶系统或羟基脲）时，其中的某些修饰不存在。我们表明DNA损伤增加了富马酸酶的酶活性，我们假设它受翻译后修饰的影响。发现在赖氨酸78和79处富马酶的琥珀酰化和泛素化，在苏氨酸122，丝氨酸124和苏氨酸126处的磷酸化以及在精氨酸239处的脱酰胺作用在功能上相关。经过同源性分析 还发现这些残基在进化上是保守的。另一方面，丝氨酸128不是进化保守的，Fum1S128D磷酸化模拟物能够帮助DNA修复。我们的分子模型是，在没有DNA损伤诱导的情况下，以及在对酶的需求减少的情况下，上述修饰抑制了胞质富马酸酶的酶促活性。在遗传毒性胁迫下，一些富马酸酶修饰被去除，某些酶被降解，而未修饰的蛋白质被合成。该报告首次证明了翻译后修饰如何影响富马酸酶在细胞中的催化和DNA修复功能。我们的分子模型是，在没有DNA损伤诱导的情况下以及当对酶的需求减少时，上述修饰抑制了胞质富马酸酶的酶促活性。在遗传毒性胁迫下，一些富马酸酶修饰被去除，某些酶被降解，而未修饰的蛋白质被合成。该报告首次证明了翻译后修饰如何影响富马酸酶在细胞中的催化和DNA修复功能。我们的分子模型是，在没有DNA损伤诱导的情况下以及当对酶的需求减少时，上述修饰抑制了胞质富马酸酶的酶促活性。在遗传毒性胁迫下，一些富马酸酶修饰被去除，某些酶被降解，而未修饰的蛋白质被合成。该报告首次证明了翻译后修饰如何影响富马酸酶在细胞中的催化和DNA修复功能。