Acetyl-CoA Synthetase Short Chain Family Member-1 (ACSS1) catalyzes the ligation of acetate and coenzyme A to generate acetyl-CoA in the mitochondria to produce ATP through the tricarboxylic acid (TCA) cycle. We recently generated an ACSS1-acetylation (Ac) mimic knock-in mouse, where lysine 635 was mutated to glutamine (K635Q), which structurally and biochemically mimics an acetylated lysine. ACSS1 enzymatic activity is regulated, at least in part, through the acetylation of lysine 635 in mice (lysine 642 in humans), a Sirtuin 3 deacetylation target. We challenged our Acss1