Project Summary Organic acidemias (OAs), such as methylmalonic acidemia (MMA), are a group of inborn errors of metabolism that typically arise from defects in the catabolism of amino- and fatty acids. OAs are difficult to treat and have multisystemic manifestations, leading to increased morbidity and mortality. Accretion of acyl-CoA species is postulated to cause intracellular toxicity. Here, we explore an alternative pathophysiological consequence of impaired acyl-CoA metabolism: the accumulation of aberrant posttranslational modifications (PTMs) that modify enzymes in critical intracellular pathways, especially during periods of increased stress. Using a mouse model that recapitulates the hepatic mitochondriopathy of MMA (Mmut-/-;TgINS-MCK-Mut) as well as MMA patient liver tissues, I surveyed PTMs in hepatic extracts with propionyl- and malonyl-lysine antibodies. I discovered widespread hyper-acylation in the MMA mice and MMA patient tissue samples compared to their respective control samples, but not in mice with Acsf3 deficiency, a disorder of acyl-CoA synthesis. Next, I prepared anti- PTM antibody columns, purified hepatic extracts from MMA and control mice, and performed mass spectrometry to characterize the PTM proteome. Excessive acylation of enzymes involved in glutathione, urea, arginine, lysine, tryptophan, valine, isoleucine, methionine, threonine, and fatty acid metabolism were detected in the MMA mice but not controls, and further validated mass spectrometry Cps1 hyperacylation via immunoprecipitation analysis and Western blotting. In parallel, we generated, via nonenzymatic acylation reactions, PTM-modified BSA targets for in vitro analyses. We purified, then assayed, SIRT1-7 deacylase activity using BSA-PTM standards to identify the SIRT(s) that most efficiently remove MMA related PTMs. Because PTMs usually inhibit enzyme function, our observations suggest that hyperacylation of key enzymes in pathways known to be dysregulated in MMA likely contributes to altered metabolism and identifies a new set of targets for therapeutic intervention.