High-throughput proteomics studies have identified several thousand acetylation sites on more than 1000
proteins. Mitochondrial
aconitase, the Krebs cycle
enzyme that converts
citrate to
isocitrate, has been identified in many of these reports. Acetylated mitochondrial
aconitase has also been identified as a target for
sirtuin 3 (SIRT3)-catalyzed deacetylation. However, the functional significance of mitochondrial
aconitase acetylation has not been determined. Using in vitro strategies, mass spectrometric analyses, and an in vivo mouse model of
obesity, we found a significant acetylation-dependent activation of
aconitase. Isolated heart mitochondria subjected to in vitro chemical acetylation with either
acetic anhydride or
acetyl-coenzyme A resulted in increased
aconitase activity that was reversed with
SIRT3 treatment. Quantitative mass spectrometry was used to measure acetylation at 21
lysine residues and revealed significant increases with both in vitro treatments. A high-fat diet (60% of kilocalories from fat) was used as an in vivo model and also showed significantly increased mitochondrial
aconitase activity without changes in
protein level. The high-fat diet also produced an increased level of
aconitase acetylation at multiple sites as measured by the quantitative mass spectrometry assays. Treatment of isolated mitochondria from these mice with
SIRT3 abolished the high-fat diet-induced activation of
aconitase and reduced acetylation. Finally, kinetic analyses found that the increase in activity was a result of increased maximal velocity, and molecular modeling suggests the potential for acetylation at K144 to perturb the tertiary structure of the
enzyme. The results of this study reveal a novel activation of mitochondrial
aconitase by acetylation.