The post-translational modification lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. Here, using stable isotope labelling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high-stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high-stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in the urea cycle, are regulated by SIRT5. Mutation of the high-stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance, which can be reversed by the overexpression of wild-type, but not succinyl-mimetic, ASS1. Therefore, lysine succinylation is functionally important in ammonia metabolism.

翻译后修饰赖氨酸琥珀酰化涉及多种代谢途径的调节。然而，由于确定高影响琥珀酰化位点的方法学困难，其生物学相关性仍然不确定。在这里，使用稳定同位素标记和数据独立的采集质谱法，我们量化了小鼠肝脏中的赖氨酸琥珀酰化化学计量。尽管赖氨酸琥珀酰化的总体化学计量较低，但仍鉴定出几个高化学计量位点，特别是在删除去琥珀酰酶 SIRT5 后。特别是，精氨基琥珀酸合酶 (ASS1)（尿素循环中的关键酶）中鉴定的多个高化学计量赖氨酸位点受 SIRT5 调节。ASS1 中的高化学计量赖氨酸突变为琥珀酰模拟谷氨酸显着降低了其酶活性。代谢组学分析证实 SIRT5 缺乏会降低肝脏中的尿素循环活性。重要的是，SIRT5 缺乏会损害氨耐受性，这种情况可以通过野生型 ASS1 的过度表达来逆转，但不能通过琥珀酰模拟物 ASS1 来逆转。因此，赖氨酸琥珀酰化在氨代谢中具有重要的功能。