Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II

摘要

class="head no_bottom_margin" id="sec1title">IntroductionAcetyl coenzyme A (AcCoA) is central to mitochondrial metabolism, providing acetyl groups to the citric acid cycle from the oxidation of carbohydrate and fat (). One way that AcCoA affects mitochondria is through N-acetylation of the ε-amino of lysine residues on mitochondrial proteins. The significance of N-acetylation is implied by the existence of a mitochondrial deacetylase, sirtuin 3 (Sirt3), which uses NAD⁺ to remove acetyl groups from lysine residues, and by the observation that Sirt3 is important in the pathology of a range of degenerative diseases, including cancer, aging, and diabetes ().Although the enzymatic basis of the deacetylation of N-acetylated lysine residues by Sirt3 is established, the mechanism by which mitochondrial AcCoA acetylates lysine residues is less certain. Although a mitochondrial N-acetyltransferase (Gcn5L1) has been proposed (), more interesting in the context of degenerative disease is the observation that mitochondrial protein is non-enzymatically N-acetylated on lysine residues by AcCoA (, , , ). The mitochondrial concentration of coenzyme A (CoA) in vivo (∼1 mM) is usually higher than AcCoA (∼100–500 μM) (), but this shifts in the presence of fatty acids with AcCoA rising (∼1 mM) and CoA falling (∼100–300 μM). Consequently, both the mitochondrial concentration of AcCoA and the AcCoA/CoA ratio alter markedly upon changes to nutrition or exercise. Whether protein lysine acetylation is a damaging consequence of relying on AcCoA for metabolism or a regulatory pathway to respond to changes in AcCoA, the AcCoA/CoA ratio or NAD⁺ is unclear. Even so, Sirt3 plays a major pathophysiological role in reversing the damaging lysine N-acetylation of mitochondrial proteins that arises from exposure to excess AcCoA.The mechanism proposed for non-enzymatic lysine acetylation was a nucleophilic attack by the side-chain amine on the acetyl carbonyl (). This direct reaction will be slow in the mitochondrial matrix (pH ∼7.8), because the pKa of a free lysine is ∼10.5. Although this rate can be enhanced by stabilization of the amine, protein cysteine residues are on average better nucleophiles (pKa ∼8.5) (). Because cysteine-containing peptides can be directly S-acetylated by AcCoA in vitro (), we postulated that S-acetylation of protein cysteine thiols would be far higher than N-acetylation of lysine amines. Here we show that S-acetylation is a frequent modification and that non-enzymatic N-acetylation of lysine residues by AcCoA occurs predominantly via a proximal S-acetylated thiol intermediate. We also show that glutathione (GSH) and glyoxalase II (Glo2), also known as hydroxyacyl glutathione hydrolase (HAGH), together can limit S-acetylation and consequently N-acetylation.