Mechanism of Nicotinamide Inhibition and Transglycosidation by Sir2 Histone/Protein Deacetylases

Abstract

Silent information regulator 2 (Sir2) enzymes catalyze NAD؉ -dependent protein/histone deacetylation, where the acetyl group from the lysine ⑀-amino group is transferred to the ADP-ribose moiety of NAD ؉ , producing nicotinamide and the novel metabolite O-acetyl-ADPribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD ؉ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Brö nsted plot of the data yielded a slope of ؉0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD ؉ analogues including ␤-2-deoxy-2-fluororibo-NAD ؉ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2-OH ribose. The observed enzyme-catalyzed formation of a labile 1-acetylated-ADP-fluororibose intermediate using ␤-2-deoxy-2-fluororibo-NAD ؉ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1 ribose to form an initial iminium adduct.The acetylation state of histones is intimately coupled to transcription, DNA repair, and replication and is governed by the competing enzymatic activities of histone acetyltransferases and histone deacetylases (reviewed in Refs. 1-3). Recently a new family of histone deacetylases has emerged and is referred to as the silent information regulator 2 (Sir2) 1 family of histone/protein deacetylases (reviewed in Refs. 4 -6) or Sirtuins (7). This family is highly conserved from prokaryotes to humans (7), and there is evidence suggesting that the scope of Sir2 activity extends beyond histone deacetylation and involves other protein targets throughout the cell. In yeast, at least five Sir2-like proteins have been identified. The founding member, yeast Sir2 (ySir2), is required for all major silenced loci (reviewed in Ref. 4). A Sir2 homologue from Salmonella enterica was shown to up-regulate acetyl-CoA synthetase, through deacetylation of a critical lysine residue (8, 9). In humans, seven Sir2 homologues have been identified to date (7). Of these seven, human SIRT2 (hSIRT2) has been identified as a cytosolic protein (10) that deacetylates ␣-tubuli...

Sir2 Protein Deacetylases: Evidence for Chemical Intermediates and Functions of a Conserved Histidine

“…These experiments were performed by varying NAD + at saturating AcH3 concentrations such that the pH dependency of k cat /K m would reflect ionizations critical for any steps from NAD + binding to nicotinamide release. Determination of HST2 WT k cat /K m values over a pH range from 5.4 to 9.4 revealed a critical ionization with a pK a value of 6.77 ± 0.06 that must be unprotonated for activity (Figure 2A) (39). Therefore, the critical ionization seen in the wild-type k cat /K m pH profile is consistent with the His-135 sidechain binding and positioning NAD + into its catalytically productive conformation.…”

“…This unusual consumption of NAD + in sirtuins (class III HDACs) with linked generation of the novel product OAADPr, has led to the idea that OAADPr possesses unique biological properties and whose cellular levels may be tightly regulated (32). Indeed, OAADPr was shown to be metabolized by a variety of cell extracts (33,34 (39,40). In addition, the 2'-hydroxyl of NAD + does not play a significant role in the first chemical step since replacement with fluorine results in only a slight decrease in the nicotinamide exchange rate (39).…”

“…The small three-fold change in the nicotinamide exchange rate observed between HST2 WT and HST2 H135A (39) suggests that the slowed catalytic step seen in the HST2 H135A k cat pH profile ( Figure 2B) occurs after nicotinamide cleavage and release, but before OAADPr and peptide release. By default, this affected step would be the 2'-hydroxyl attack of the α-1'-O-alkylamidate intermediate, which is expedited in wild-type by His-135 functioning as the general base, but is impaired in HST2 H135A.…”

“…In the initial chemical step, nicotinamide is released upon formation of an enzyme:ADP-ribose:acetylated protein intermediate, which has been postulated to be an α-1'-O-alkylamidate (Scheme 1) (28)(29)(30). One line of evidence for an α-1'-O-alkylamidate is the ability of nicotinamide, a potent product inhibitor (15,16,(38)(39)(40), to rapidly react with the enzyme:ADP-ribose:acetylated protein intermediate, regenerating NAD + and acetylated protein by a process dubbed the nicotinamide exchange (or transglycosidation) reaction (39,40). In addition, the 2'-hydroxyl of NAD + does not play a significant role in the first chemical step since replacement with fluorine results in only a slight decrease in the nicotinamide exchange rate (39).…”

“…One line of evidence for an α-1'-O-alkylamidate is the ability of nicotinamide, a potent product inhibitor (15,16,(38)(39)(40), to rapidly react with the enzyme:ADP-ribose:acetylated protein intermediate, regenerating NAD + and acetylated protein by a process dubbed the nicotinamide exchange (or transglycosidation) reaction (39,40). In addition, the 2'-hydroxyl of NAD + does not play a significant role in the first chemical step since replacement with fluorine results in only a slight decrease in the nicotinamide exchange rate (39). This suggested that the initial step occurs exclusively about the 1'-position and that the acetylated-lysine carbonyl oxygen acts as a nucleophile directly displacing nicotinamide.…”