Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation

J Biol Chem. 2016 Dec 2;291(49):25398-25410. doi: 10.1074/jbc.M116.754655. Epub 2016 Oct 18.


The sirtuin family of proteins catalyze the NAD+-dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1-7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD+ and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn2+ release from the conserved sirtuin Zn2+-tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD+ and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn2+, consistent with S-nitrosation of the Zn2+-tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment.

Keywords: acetylation; allosteric regulation; enzyme inactivation; inhibition mechanism; nicotinamide adenine dinucleotide (NAD); nitric oxide; nitrosation; nitrosylation; sirtuin 1 (SIRT1); zinc.

MeSH terms

  • Animals
  • Cattle
  • Cysteine / chemistry
  • Cysteine / metabolism
  • Enzyme Stability
  • Humans
  • NAD / chemistry*
  • NAD / metabolism
  • Nitrosation
  • S-Nitrosoglutathione / chemistry*
  • Sirtuin 1 / chemistry*
  • Sirtuin 1 / metabolism
  • Zinc / chemistry*
  • Zinc / metabolism


  • NAD
  • S-Nitrosoglutathione
  • SIRT1 protein, human
  • Sirtuin 1
  • Zinc
  • Cysteine

Associated data

  • PDB/4ZZJ
  • PDB/4KXQ