Regulation of urea cycle by reversible high stoichiometry lysine succinylation

Abstract 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. In the present study, using stable isotope labeling 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 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 and reduces locomotor and exploratory activity in male mice upon high-ammonium diet feeding. Therefore, lysine succinylation is functionally important in ammonia metabolism.