Mechanism-Based Inhibition of Sir2 Deacetylases by Thioacetyl-Lysine Peptide

Abstract

Sir2 protein deacetylases (or sirtuins) catalyze NAD+-dependent conversion of epsilon-amino-acetylated lysine residues to deacetylated lysine, nicotinamide, and 2'-O-acetyl-ADP-ribose. Small-molecule modulation of sirtuin activity might treat age-associated diseases, such as type II diabetes, obesity, and neurodegenerative disorders. Here, we have evaluated the mechanisms of sirtuin inhibition of histone peptides containing thioacetyl or mono-, di-, and trifluoroacetyl groups at the epsilon-amino of lysine. Although all substituted peptides yielded inhibition of the deacetylation reaction, the thioacetyl-lysine peptide exhibited exceptionally potent inhibition of sirtuins Sirt1, Sirt2, Sirt3, and Hst2. Using Hst2 as a representative sirtuin, the trifluoroacetyl-lysine peptide displayed competitive inhibition with acetyl-lysine substrate and yielded an inhibition constant (Kis) of 4.8 microM, similar to its Kd value of 3.3 microM. In contrast, inhibition by thioacetyl-lysine peptide yielded an inhibition constant (Kis) of 0.017 microM, 280-fold lower than its Kd value of 4.7 microM. Examination of thioacetyl-lysine peptide as an alternative sirtuin substrate revealed conserved production of deacetylated peptide and 1'-SH-2'-O-acetyl-ADP-ribose. Pre-steady-state and steady-state analysis of the thioacetyl-lysine peptide showed rapid nicotinamide formation (4.5 s-1) but slow overall turnover (0.0024 s-1), indicating that the reaction stalled at an intermediate after nicotinamide formation. Mass spectral analysis yielded a novel species (m/z 1754.3) that is consistent with an ADP-ribose-peptidyl adduct (1'-S-alkylamidate) as the stalled intermediate. Additional experiments involving solvent isotope effects, general base mutational analysis, and density functional calculations are consistent with impaired 2'-hydroxyl attack on the ADP-ribose-peptidyl intermediate. These results have implications for the development of mechanism-based inhibitors of Sir2 deacetylases.

The Bicyclic Intermediate Structure Provides Insights into the Desuccinylation Mechanism of Human Sirtuin 5 (SIRT5)

“…Comparison between SIRT5-Bicyclic Intermediate and SIRT3-Alkylamidate Intermediate-Kinetic studies and mass spectrometry have demonstrated the existence of the alkylamidate and 1Ј,2Ј-cyclic intermediates (18,19 (21). When comparing the SIRT5-bicyclic intermediate structure with the SIRT3-alkylamidate intermediate structure (Fig.…”

“…The absolutely conserved histidine residue among sirtuins serves as a general base to deprotonate the 2Ј-OH directly or indirectly through the deprotonation of the 3Ј-OH to attack the 1Ј-O-alkylamidate. Kinetic and mass spectrometry experiments suggested the existence of the alkylamidate and the bicyclic intermediates (18,19). Using a mechanism-based inhibitor (a thioacetyl-lysine peptide), the S-alkylamidate intermediate was captured in Sir2Tm and SIRT3 crystals (20,21).…”

Sirtuin Catalysis and Regulation

“…Utilization of acetyllysine analogs further demonstrated the existence of the alkylamidate intermediate. Thioacetyllysine-and acetylazalysinecontaining peptides form stalled alkylamidate intermediates when used as sirtuin substrates (19,20). Upon formation of the alkylamidate intermediate, the 2Ј-hydroxyl group of the NAD ϩ ribose is activated by a conserved active-site histidine (supplemental Fig.…”

Genomewide Screen for Negative Regulators of Sirtuin Activity inSaccharomyces cerevisiaeReveals 40 Loci and Links to Metabolism

“…Work in a variety of systems has shown that they play roles in many key cellular processes including gene regulation, aging, cell survival, metabolic control, and DNA repair (Brachmann et al 1995;Kaeberlein et al 1999;Lin et al 2000;Langley et al 2002). Small molecule inhibitors and activators of sirtuin activity have received considerable attention recently as potential therapeutic agents for aging-associated diseases including Parkinson's disease and type II diabetes (Smith and Denu 2007). Thus, there is substantial general interest in understanding how this family of enzymes is regulated.…”