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• W2003155232 abstract "Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine ϵ-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose. 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 Brönsted 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 β-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 β-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. Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine ϵ-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose. 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 Brönsted 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 β-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 β-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. 1Kouzarides T. EMBO J. 2000; 19: 1176-1179Crossref PubMed Scopus (1010) Google Scholar, 2Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1624) Google Scholar, 3Grozinger C.M. Schreiber S.L. Chem. Biol. 2002; 9: 3-16Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). Recently a new family of histone deacetylases has emerged and is referred to as the silent information regulator 2 (Sir2) 1The abbreviations used are: Sir2silent information regulator 2OAADPrO-acetyl-ADP-riboseHPLChigh pressure liquid chromatography2′-ribo-FNAD+2′-deoxy-2′-fluoro-NAD+thio-NAD+thionicotinamide adenine dinucleotide3-Ac-PAD+3-acetylpyridine adenine dinucleotide3-h-PAD+3-hydroxypyridine adenine dinucleotidewtwild typeDTTdithiothreitolPBSphosphate-buffered saline. family of histone/protein deacetylases (reviewed in Refs. 4Moazed D. Curr. Opin. Cell Biol. 2001; 13: 232-238Crossref PubMed Scopus (150) Google Scholar, 5Gasser S.M. Cockell M.M. Gene (Amst.). 2001; 279: 1-16Crossref PubMed Scopus (229) Google Scholar, 6Denu J.M. Trends Biochem. Sci. 2003; 28: 41-48Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) or Sirtuins (7Frye R.A. Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1174) Google Scholar). This family is highly conserved from prokaryotes to humans (7Frye R.A. Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1174) Google Scholar), 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. 4Moazed D. Curr. Opin. Cell Biol. 2001; 13: 232-238Crossref PubMed Scopus (150) Google Scholar). A Sir2 homologue from Salmonella enterica was shown to up-regulate acetyl-CoA synthetase, through deacetylation of a critical lysine residue (8Starai V.J. Celic I. Cole R.N. Boeke J.D. Escalante-Semerena J.C. Science. 2002; 298: 2390-2392Crossref PubMed Scopus (478) Google Scholar, 9Starai V.J. Takahashi H. Boeke J.D. Escalante-Semerena J.C. Genetics. 2003; 163: 545-555Crossref PubMed Google Scholar). In humans, seven Sir2 homologues have been identified to date (7Frye R.A. Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1174) Google Scholar). Of these seven, human SIRT2 (hSIRT2) has been identified as a cytosolic protein (10Afshar G. Murnane J.P. Gene (Amst.). 1999; 234: 161-168Crossref PubMed Scopus (116) Google Scholar) that deacetylates α-tubulin at lysine 40, both in vitro and in vivo (11North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. Mol. Cell. 2003; 11: 437-444Abstract Full Text Full Text PDF PubMed Scopus (1251) Google Scholar). Recent work has also shown that hSIRT3 is localized to the mitochondria (12Schwer B. North B.J. Frye R.A. Ott M. Verdin E. J. Cell Biol. 2002; 158: 647-657Crossref PubMed Scopus (465) Google Scholar, 13Onyango P. Celic I. McCaffery J.M. Boeke J.D. Feinberg A.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13653-13658Crossref PubMed Scopus (444) Google Scholar). The nuclear Sir2 homologue, SIRT1, has been reported to regulate the p53 tumor suppressor by deacetylation, resulting in inhibition of the p53-dependent apoptotic pathway (14Vaziri H. Dessain S.K. Ng Eaton E. Imai S.I. Frye R.A. Pandita T.K. Guarente L. Weinberg R.A. Cell. 2001; 107: 149-159Abstract Full Text Full Text PDF PubMed Scopus (2316) Google Scholar, 15Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1902) Google Scholar). silent information regulator 2 O-acetyl-ADP-ribose high pressure liquid chromatography 2′-deoxy-2′-fluoro-NAD+ thionicotinamide adenine dinucleotide 3-acetylpyridine adenine dinucleotide 3-hydroxypyridine adenine dinucleotide wild type dithiothreitol phosphate-buffered saline. Class 1 and 2 histone deacetylases catalyze the deacetylation of histones/proteins to generate free acetate and the deacetylated protein. Based on sequence homology to a metalloprotease-like enzyme from the hyperthermophilic bacterium Aquifex aeolicus, the mechanism involves activation of a water molecule by a zinc atom and two aspartate/histidine ion pairs in the active site (16Finnin M.S. Donigian J.R. Cohen A. Richon V.M. Rifkind R.A. Marks P.A. Breslow R. Pavletich N.P. Nature. 1999; 401: 188-193Crossref PubMed Scopus (1509) Google Scholar). In contrast, Sir2-like enzymes, which constitute class 3 histone deacetylases, require NAD+ for catalytic activity (17Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Crossref PubMed Scopus (624) Google Scholar, 18Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar, 19Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2817) Google Scholar, 20Landry J. Sutton A. Tafrov S.T. Heller R.C. Stebbins J. Pillus L. Sternglanz R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5807-5811Crossref PubMed Scopus (820) Google Scholar), and deacetylation of substrate is tightly coupled to the formation of a novel compound, O-acetyl-ADP-ribose (OAADPr) (18Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar, 21Tanny J.C. Moazed D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 415-420Crossref PubMed Scopus (225) Google Scholar). Based on several lines of evidence (22Jackson M.D. Denu J.M. J. Biol. Chem. 2002; 277: 18535-18544Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 23Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Crossref PubMed Scopus (261) Google Scholar, 24Chang J.H. Kim H.C. Hwang K.Y. Lee J.W. Jackson S.P. Bell S.D. Cho Y. J. Biol. Chem. 2002; 277: 34489-34498Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), the initial enzymatic product is 2′-O-acetyl-ADP-ribose (2′-OAADPr), which subsequently equilibrates with 3′-O-acetyl-ADP-ribose (3′-OAADPr) in solution through a nonenzymatic intramolecular transesterification reaction. Although the physiological function of OAADPr is unclear, microinjection of OAADPr results in a delay/block in oocyte maturation and in a delay/block of embryo cell division (25Borra M.T. O'Neill F.J. Jackson M.D. Marshall B. Verdin E. Foltz K.R. Denu J.M. J. Biol. Chem. 2002; 277: 12632-12641Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Enzymatic activities that metabolize OAADPr in cell extracts have been described (26Rafty L.A. Schmidt M.T. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2002; 277: 47114-47122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), providing further evidence for an important role in cell signaling or metabolism. In yeast, several recent reports (17Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Crossref PubMed Scopus (624) Google Scholar, 27Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Cohen H. Lin S.S. Manchester J.K. Gordon J.I. Sinclair D.A. J. Biol. Chem. 2002; 277: 18881-18890Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 28Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar, 29Sandmeier J.J. Celic I. Boeke J.D. Smith J.S. Genetics. 2002; 160: 877-889Crossref PubMed Google Scholar) have implicated a nuclear NAD+ salvage pathway as a regulatory control point for ySir2. This salvage pathway conserves the pyridine ring of nicotinic acid or nicotinamide to regenerate β-NAD+. Enzymes involved in this pathway influence ySir2-dependent gene silencing. Mutation of the NAD+ salvage pathway gene NPT1 (nicotinate phosphoribosyltransferase) results in silencing defects (29Sandmeier J.J. Celic I. Boeke J.D. Smith J.S. Genetics. 2002; 160: 877-889Crossref PubMed Google Scholar), whereas a single extra copy of the PNC1 gene (nicotinamidase) is sufficient to increase ySir2-dependent silencing (28Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar). Additional copies of NPT1 led to increases in Sir2-dependent silencing and extended yeast replicative life span (27Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Cohen H. Lin S.S. Manchester J.K. Gordon J.I. Sinclair D.A. J. Biol. Chem. 2002; 277: 18881-18890Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). How these pathway intermediates (nicotinamide metabolites) may affect directly Sir2-dependent deacetylation is not clear. However, nicotinamide has been suggested as an in vivo regulator of Sir2 function (6Denu J.M. Trends Biochem. Sci. 2003; 28: 41-48Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 28Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar), as physiological concentrations of nicotinamide inhibited ySir2, HST2, and human SIRT1 (hSIRT1) in vitro (28Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar, 30Landry J. Slama J.T. Sternglanz R. Biochem. Biophys. Res. Commun. 2000; 278: 685-690Crossref PubMed Scopus (221) Google Scholar), whereas exogenous nicotinamide added to yeast cells decreased gene silencing and accelerated aging (28Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar), hallmarks of Sir2-deficient yeast. Although nicotinamide has been shown to be a potent inhibitor of several Sir2 homologues (28Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar, 30Landry J. Slama J.T. Sternglanz R. Biochem. Biophys. Res. Commun. 2000; 278: 685-690Crossref PubMed Scopus (221) Google Scholar), the mechanism of inhibition has not been established. This paper focuses on three critical topics as follows: the mechanism of nicotinamide inhibition through the measurement of the efficiency by which nicotinamide/pyridine analogues can serve as substrates for transglycosidation; the nature of the transition state for transglycosidation; and the function of the 2′-hydroxyl of the nicotinamide ribose in the catalytic mechanism utilizing NAD+ analogues and a catalytic mutant (His-to-Ala). Reagents—All chemicals were of the highest purity commercially available, were purchased from Sigma, Aldrich, or Fisher, and were used without further purification. Synthetic 11-mer H3 peptide and acetylated H3 peptide (corresponding to the 10 amino acid residues surrounding lysine 14 of histone H3; H2N-KSTGGK(Ac)APRKQCONH2) were purchased from SynPep Corporation (Dublin, CA), 3H-Labeled acetyl H3-peptide was prepared as reported previously (22Jackson M.D. Denu J.M. J. Biol. Chem. 2002; 277: 18535-18544Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). [adenylate-32P]NAD+ was purchased from ICN (Costa Mesa, CA). HST2 mutant H135A was a generous gift from Rolf Sternglanz (State University of New York, Stony Brook). 2′-Ribo-FNAD+ and 2′-deoxy-ribo-NAD+ were synthesized as described previously (31Sleath P.R. Handlon A.L. Oppenheimer N.J. J. Org. Chem. 1991; 56: 3608-3618Crossref Scopus (27) Google Scholar). Expression and Purification of His-tagged HST2, HST2 Mutant H135A, hSIRT2, and ySir2—The procedure used in the expression and purification of histidine-tagged full-length HST2 has been reported previously (22Jackson M.D. Denu J.M. J. Biol. Chem. 2002; 277: 18535-18544Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) with only minor changes and was applied to the purification of the Sir2 enzymes. Fractions containing protein greater than 90% purity, based upon densitometry, were pooled, concentrated, and dialyzed in 50 mm Tris (pH 7.5, 37 °C) or PBS (pH 7.3), 10% glycerol, and 1 mm DTT. For yeast Sir2 (ySir2), 800 mm NaCl was also included in the final dialysis to prevent precipitation of protein. Enzyme concentrations were determined using the method of Bradford (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar), and the stock solutions were stored at -25 °C until needed. Charcoal Binding Assays—General enzymatic reactions contained 3H-acetylated H3 peptide substrate, NAD+, and a NAD+-dependent Sir2-like class III protein/histone deacetylase to form the product 3H-labeled OAADPr. To 70-μl reaction volumes was added 50 μl of charcoal slurry (1 volume of activated charcoal to 2 volumes of 2.0 m glycine buffer, pH 9.5) to terminate the reaction at the desired time points. The mixture was heated at 95 °C for 1 h and then centrifuged for 1 min at 7000 × g. A total of 90 μl of the supernatant was removed and added to 50 μl of fresh charcoal slurry, centrifuged again for 1 min, and 100 μl of the supernatant removed and added to a clean tube. This was centrifuged for 5 min, and 75 μl removed and quantified by liquid scintillation counting. This assay effectively measures the amount of 3H-labeled OAADPr in the form of free acetate. Both OAADPr and acetylated H3 peptide bind to the activated charcoal. However, under these conditions the 3H-labeled acetate from OAADPr is hydrolyzed and remains in the supernatant. Activated charcoal immediately stops the enzymatic reaction at all pH and temperature values tested. Heating and high pH is necessary only to hydrolyze acetate from OAADPr (data not shown). Amides, represented by unreacted peptide, are not hydrolyzed under these conditions (data not shown). Nicotinamide Exchange Reactions—General exchange reactions for HST2, HST2 mutant H135A, hSIRT2, and ySir2 were performed in 50-μl volumes containing 500 μm NAD+, 300 μm acetylated H3 peptide, 1 mm DTT, and buffer (PBS, pH 7.3, 37 °C, or 50 mm Tris-Cl, pH 7.5, 37 °C, as noted), 0.2 μm enzyme, and nicotinamide concentrations ranging from 3 μm to 1 mm containing [carbonyl-14C]nicotinamide (Sigma N-2142, 52.8 mCi/mmol). Reactions were initiated via addition of enzyme, incubated at 37 °C for 5-8 min, and quenched with neat trifluoroacetic acid (to a final concentration of 1%). Linearity of rates was confirmed by measuring exchange over time courses up to 30 min. After quenching, 5 μl of each reaction was spotted to Whatman aluminum-backed Silica Gel TLC plates (Fisher) and placed in a development tank pre-equilibrated with 80:20 ethanol, 2.5 m ammonium acetate for 1 h (20Landry J. Sutton A. Tafrov S.T. Heller R.C. Stebbins J. Pillus L. Sternglanz R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5807-5811Crossref PubMed Scopus (820) Google Scholar). Plates were air-dried and exposed to a PhosphorImaging screen (Bio-Rad Molecular Imaging Screen-CS) for 24 h and then read with Bio-Rad GS-525 Molecular Imaging System and Molecular Analyst software (version 2.1.2). The percentage of nicotinamide exchanged was determined by measuring the densities of the [carbonyl-14C]nicotinamide (Rf 0.80) and enzymatically created 14C-labeled NAD+ (Rf 0.27). Enzymatic exchange rates were fitted to the Michaelis-Menten equation (Equation 1) (Kaleidagraph, Synergy Software, Reading, PA). Reported values are the average of at least three independent experiments.νo=(kcat·[S])/(Km+[S])(Eq. 1) Nicotinamide Inhibition Curves—Nicotinamide inhibition reactions for both HST2 and hSIRT2 were performed in 80-μl volumes with 500 μm NAD+, 300 μm3H-acetylated H3 peptide, 1 mm DTT, 50 mm Tris-Cl (pH 7.5, 37 °C), 0.5 μm enzyme, and nicotinamide concentrations ranging from 3 μm to 1 mm. Reactions were initiated by addition of enzyme, incubated at 37 °C for 5-8 min, and quenched with activated charcoal. Inhibition was determined by measuring the forward rate using the charcoal-binding assay (described above). Forward rates were fitted to Equation 2 using Kaleidagraph software. Reported values are the average of at least three independent experiments.νapp=νmax(1-([I]/(Ki(app)+[I])))(Eq. 2) Exchange Rates of Pyridine Derivatives for Use in Brönsted Analyses—Exchange reactions were performed in 70-μl volumes containing 500 μm NAD+, 300 μm acetylated H3 peptide, 1 mm DTT, 50 mm Tris-Cl (pH 7.5, 37 °C), 0.2-1.0 μm HST2, and pyridine analogue concentrations ranging from 0.4 to 100 mm. The pH values of inhibitor stock solutions were checked for consistency. Reactions were initiated via addition of enzyme, incubated at 37 °C, and quenched with neat trifluoroacetic acid (to a final concentration of 1%). Quenched reactions were analyzed by HPLC. Two analytical C-18 columns (Vydac, 4.6 × 250 mm, 10 μm) were connected in tandem and run at 1 ml/min in 100% buffer A (0.05% trifluoroacetic acid/water) for 1 min and then ramped to 20% buffer B (0.02% trifluoroacetic acid/acetonitrile) for over 20 min. NAD+ eluted from the column at 13 min, and the corresponding NAD+ analogue made in the exchange reaction eluted 1-3 min later. Rates were determined by integrating the peaks and determining the amount of NAD+ converted to its analogue. Reactions were quenched at 1, 2, and 3 min, and rates were determined by using linear least squares regression with Kaleidagraph software. Corresponding rates were then fitted to the Michaelis-Menten equation (Equation 1). The log of the rate for pyridine analogue exchange (Vmax) was plotted against pKa of the ring nitrogen of the pyridine analogue in the Brönsted plot and fitted using linear least squares regression (Kaleidagraph software). TLC Conditions for the Analysis of Thionicotinamide and 3-Hydroxypyridine Inhibition Reactions—Inhibition reactions were performed in 60-μl volumes with 50 μm32P-labeled NAD+ (ICN 37025, adenylate-32P), 225 μm acetylated H3 peptide, 1 mm DTT, 50 mm inhibitor, 4.2 μm HST2, in 50 mm sodium phosphate buffer (pH 7.5, 37 °C). Nicotinamide was used as a control. Thionicotinamide reactions were run in 6.8% dimethyl sulfoxide (Me2SO). Reactions were run for 10 min at 37 °C and quenched with neat trifluoroacetic acid to a final concentration of 1%. A total of 2 μl of each reaction was spotted onto Whatman aluminum-backed silica gel TLC plates and placed in a development tank pre-equilibrated with 70:30 ethanol, 2.5 m ammonium acetate for 4 h. The plate was air-dried, exposed to a β-imaging screen (Bio-Rad Molecular Imaging Screen-BI) for 3 h, and read as described above. Under these conditions NAD+ has an Rf of 0.41, ADPr an Rf of 0.68, and OAADPr an Rf of 0.75. Charcoal Binding Method Used for the Analysis of Pyridine Analogue Inhibition Reactions—Inhibition reactions were performed in 80-μl volumes with 50 μm NAD+, 225 μm3H-acetylated H3 peptide, 1 mm DTT, 0.4 μm HST2, inhibitor concentrations spanning 1-30 mm, and in 50 mm sodium phosphate buffer (pH 7.5, 37 °C). Reactions were initiated by addition of enzyme, incubated at 37 °C for 5-8 min, and quenched with activated charcoal. Inhibition was determined by measuring the forward rate using the charcoal-binding assay (described above). Reactions using thionicotinamide contained 6.8% Me2SO and were compared with uninhibited reactions also in 6.8% Me2SO as a control. Reported values are the average of at least three independent experiments. Use of 2′-Deoxy-2′-fluororibo-NAD+and in Nicotinamide Exchange Reactions Catalyzed by HST2, HST2 Mutant H135A, and hSirT2—At fixed [nicotinamide], exchange reactions using 2′-ribo-FNAD+ consisted of either 80-100 μm wt HST2 or HST2 mutant H135A or 50 μm hSIRT2, 800 μm 2′-ribo-FNAD+, 300 μm AcH3 peptide, 50 μm nicotinamide (containing 14C-labeled nicotinamide), 1 mm DTT, in phosphate-buffered saline (pH 7.3) at 37 °C. Reactions were initiated via the addition of enzyme and incubated for the indicated times at 37 °C. Reactions were quenched via the addition of neat trifluoroacetic acid to a final concentration of 1%, and 6-μl aliquots were analyzed by TLC as mentioned previously. To obtain the apparent Vmax value for nicotinamide exchange with 2′-ribo-FNAD+, reactions were carried out at 37 °C for 7.5 min and contained the following components: 7.8 μm hSIRT2 or 79 μm HST2, 1 mm DTT, 300 μm AcH3 peptide, 1.91 mm dFNAD in PBS (pH 7.3, 37 °C), and nicotinamide concentrations ranging from 50 to 2000 μm. Samples were incubated at 37 °C for 5 min prior to the addition of enzyme. Reactions were terminated via the addition of neat trifluoroacetic acid to a final concentration of 1% and were analyzed by HPLC and liquid scintillation counting as described before. Control reactions containing the varied range of [nicotinamide] but lacking enzyme were treated in the same manner to determine the extent of nonenzymatic incorporation of exogenous nicotinamide. The initial rate of nicotinamide exchange was determined by subtracting the amount of nicotinamide incorporation from the controls from the enzymatic reactions. The complete data set was then fitted to the Michaelis-Menten equation to obtain the apparent Vmax for base exchange. The 2′-deoxy-ribo-NAD+ analogue was analyzed similarly to that of 2′-deoxy-2′-fluororibo-NAD+ except where noted in the legend of Fig. 5B. Utilization of 2′-Deoxy-2′-fluororibo-NAD+ as a Substrate in Forward Reactions Catalyzed by Wild-type HST2—Reactions were performed in triplicate and consisted of 100 μm HST2, 1 mm DTT, 500 μm [3H]AcH3 peptide, 800 μm 2′-ribo-FNAD+ in phosphate-buffered saline (pH 7.3, 37 °C). The reactions and controls were incubated at 37 °C from 0.25 to 30 min and quenched via the addition of charcoal slurry, either at pH 7 or pH 9.5. The transfer of [3H]acetate from substrate to the ADP-ribose moiety of 2′-ribo-FNAD+, in the form of free acetate, was determined using the charcoal-binding assay mentioned previously. Control reactions contained either no enzyme or 1% SDS and enzyme. Free acetate does not bind to charcoal at either pH 7 or pH 9.5, whereas [3H]AcH3 peptide binds under both conditions and does not liberate free acetate, even under the pH 9.5 conditions. As with the normal product 2′-OAADPr generated by using NAD+, the observed acetylated intermediate with 2′-ribo-FNAD+ was measured as free acetate production under hydrolytic quenching. Under nonhydrolytic quenching (pH 7), this product bound to activated charcoal. Enzymatic Synthesis of [carbonyl-14C]2′-ribo-FNAD+—A single reaction (1 ml) containing 100 μm HST2, 1 mm DTT, 500 μm acetylated H3 peptide, 800 μm 2′-ribo-FNAD+, and 100 μm [carbamoyl-14C]nicotinamide in PBS (pH 7.3, 37 °C) was incubated at 37 °C for 30 min. The reaction was terminated by the addition of neat trifluoroacetic acid to a final concentration of 1%, and the [carbonyl-14C]2′-FNAD+ was purified by HPLC as described previously (22Jackson M.D. Denu J.M. J. Biol. Chem. 2002; 277: 18535-18544Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The peak corresponding to 2′-FNAD+ was collected, lyophilized, and suspended in a small volume (100 μl) of water. The concentration of [carbonyl-14C]2′-FNAD+ was determined spectrophotometrically using the extinction coefficient of 18.1 mm-1 cm-1 at 260 nm. Determination of the Rate of Nicotinamide Release Using 2′-ribo-FNAD+and Wild-type HST2—Reactions were performed in duplicate and consisted of 80 μm HST2, 1 mm DTT, 500 μm AcH3 peptide, 800 μm 2′-ribo-FNAD+ (labeled with [14C]nicotinamide) in phosphate-buffered saline (pH 7.3, 37 °C). The reactions were incubated at 37 °C for the indicated time intervals and quenched via the addition of neat trifluoroacetic acid to a final concentration of 1%. Reactions were then analyzed by HPLC as described previously. The extent of nicotinamide liberation was determined by liquid scintillation counting and correlated to nicotinamide concentrations in the reactions. Linear data points were plotted using linear least squares, and the rate of nicotinamide release was calculated from the slope of the graph. The following experiments have focused on the following three topics: the origin of nicotinamide noncompetitive inhibition; the nature of the transition state for formation of the nicotinamide-ribosyl bond; and the utilization of NAD+ analogues to probe the mechanism of deacetylation catalyzed by Sir2-like enzymes. Mechanism of Nicotinamide Inhibition—Several recent reports (17Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Crossref PubMed Scopus (624) Google Scholar, 27Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Cohen H. Lin S.S. Manchester J.K. Gordon J.I. Sinclair D.A. J. Biol. Chem. 2002; 277: 18881-18890Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 28Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar) have implicated a nuclear NAD+ salvage pathway in the regulation of ySir2 function. Nicotinamide, a key salvage intermediate, has been suggested as an in vivo negative regulator of ySir2 activity (6Denu J.M. Trends Biochem. Sci" @default.
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• W2003155232 title "Mechanism of Nicotinamide Inhibition and Transglycosidation by Sir2 Histone/Protein Deacetylases" @default.
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