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

Abstract

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.