Progesterone receptor membrane component 1 facilitates Ca2+ signal amplification between endosomes and the endoplasmic reticulum.

Membrane contact sites (MCSs) between endosomes and the endoplasmic reticulum (ER) are thought to act as specialized trigger zones for Ca2+ signaling, where local Ca2+ released via endolysosomal ion channels is amplified by ER Ca2+-sensitive Ca2+ channels into global Ca2+ signals. Such amplification is integral to the action of the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, functional regulators of inter-organellar Ca2+ crosstalk between endosomes and the ER remain poorly defined. Here, we identify progesterone receptor membrane component 1 (PGRMC1), an ER transmembrane protein that undergoes a unique heme-dependent dimerization, as an interactor of the endosomal two pore channel, TPC1. NAADP-dependent Ca2+ signals were potentiated by PGRMC1 overexpression through enhanced functional coupling between endosomal and ER Ca2+ stores and inhibited upon PGRMC1 knockdown. Point mutants in PGMRC1 or pharmacological manipulations that reduced its interaction with TPC1 were without effect. PGRMC1 therefore serves as a TPC1 interactor that regulates ER-endosomal coupling with functional implications for cellular Ca2+ dynamics and potentially the distribution of heme.

Convergent activation of two-pore channels mediated by the NAADP-binding proteins JPT2 and LSM12.

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) evokes calcium ion (Ca2+) release from endosomes and lysosomes by activating two-pore channels (TPCs) on these organelles. Rather than directly binding to TPCs, NAADP associates with proteins that indirectly confer NAADP sensitivity to the TPC complex. We investigated whether and how the NAADP-binding proteins Jupiter microtubule-associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12) contributed to NAADP-TPC-Ca2+ signaling in human cells. Biochemical and functional analyses revealed that recombinant JPT2 and LSM12 both bound to NAADP with high affinity and that endogenous JPT2 and LSM12 independently associated with TPC1 and TPC2. On the basis of knockout and rescue analyses, both NAADP-binding proteins were required to support NAADP-evoked Ca2+ signaling and contributed to endolysosomal trafficking of pseudotyped coronavirus particles. These data reveal that the NAADP-binding proteins JPT2 and LSM12 convergently regulate NAADP-evoked Ca2+ release and function through TPCs.

Synthesis and biological evaluation of novel photo-clickable adenosine and cyclic ADP-ribose analogs: 8-N3-2'-O-propargyladenosine and 8-N3-2'-O-propargyl-cADPR.

A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2'-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N3-2'-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5'-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N3-2'-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N3-2'-O-propargyl cyclic ADP-ribose. Photo-clickable cyclic-ADP-ribose analog was envisioned as a probe to label cyclic ADP-ribose binding proteins. The monofunctional 8-N3-cADPR has previously been shown to be an antagonist of cADPR-induced calcium release [T.F. Walseth et. al., J. Biol. Chem (1993) 268, 26686-26691]. 2'-O-propargyl-cADPR was recognized as an agonist which elicited Ca2+ release when added at low concentration to sea urchin egg homogenates. The bifunctional 8-N3-2'-O-propargyl cyclic ADP-ribose did not elicit Ca2+ release at low concentration or impact cyclic ADP-ribose mediated Ca2+ release either when added to sea urchin egg homogenates or when microinjected into cultured human U2OS cells. The photo-clickable adenosine will none-the-less be a useful scaffold for synthesizing photo-clickable probes for identifying proteins that interact with a variety of adenosine nucleotides.

NAADP-binding proteins find their identity.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from endosomes and lysosomes by activating ion channels called two-pore channels (TPCs). However, no NAADP-binding site has been identified on TPCs. Rather, NAADP activates TPCs indirectly by engaging NAADP-binding proteins (NAADP-BPs) that form part of the TPC complex. After a decade of searching, two different NAADP-BPs were recently identified: Jupiter microtubule associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12). These discoveries bridge the gap between NAADP generation and NAADP activation of TPCs, providing new opportunity to understand and manipulate the NAADP-signaling pathway. The unmasking of these NAADP-BPs will catalyze future studies to define the molecular choreography of NAADP action.

Essential requirement for JPT2 in NAADP-evoked Ca2+ signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.

Chemo-enzymatic synthesis of adenine substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs.

Nicotinamide adenine dinucleotide phosphate (NADP) is an indispensable metabolic co-substrate and nicotinic acid adenine dinucleotide phosphate (NAADP) is an important Ca2+ releasing intracellular second messenger. Exploration of the NADP and NAADP interactome often requires the synthesis of NADP derivatives substituted on the adenosine nucleoside. The introduction of the 2'-phosphate of NADP makes the synthesis of substituted NADP derivatives difficult. We have employed recombinant human NAD kinase expressed in E. coli as an enzymatic reagent to convert readily available synthetic NAD derivatives to NADP analogs, which were subsequently transformed into NAADP derivatives using enzyme catalyzed pyridine base exchange. 8-Ethynyl-NADP, 8-ethynyl-NAADP and 5-N3-8-ethynyl-NAADP were synthesized starting from a protected 8-ethynyladenosine using a combination of chemical and enzymatic steps and the NAADP derivatives shown to be recognized by the sea urchin NAADP receptor at low concentration. Our methodology will enable researchers to produce mono- and bi-substituted NADP and NAADP analogs that can be applied in proteomic studies to identify NADP and NAADP binding proteins.

The synthesis and characterization of a clickable-photoactive NAADP analog active in human cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing second messenger which triggers Ca2+ release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca2+ release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This 'all-in-one-clickable' NAADP (AIOC-NAADP) elicited Ca2+ release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca2+ release. In mammalian cell homogenates, incubation with low concentrations of [32P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [32P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [32P]5-N3-NAADP.

5-Azido-8-ethynyl-NAADP: A bifunctional, clickable photoaffinity probe for the identification of NAADP receptors.

Nicotinic acid adenine dinucleotide phosphate is an evolutionarily conserved second messenger, which mobilizes Ca2+ from acidic stores. The molecular identity of the NAADP receptor has yet to be defined. In pursuit of isolating and identifying NAADP-binding proteins, we synthesized and characterized a bifunctional probe that incorporates both a photoactivatable crosslinking azido moiety at the 5-position of the nicotinic ring and a 'clickable' ethynyl moiety to the 8-adenosyl position in NAADP. Microinjection of this 5N3-8-ethynyl-NAADP into cultured U2OS cells induced robust Ca2+ responses. Higher concentrations of 5N3-8-ethynyl were required to elicit Ca2+ release or displace 32P-NAADP in radioligand binding experiments in sea urchin egg homogenates. In human cell extracts, incubation of 32P-5N3-8-ethynyl-NAADP followed by UV irradiation resulted in selective labeling of 23 kDa and 35 kDa proteins and photolabeling of these proteins was prevented when incubated in the presence of unlabeled NAADP. Compared to the monofunctional 32P-5N3-NAADP, the clickable 32P-5N3-8-ethynyl-NAADP demonstrated less labeling of the 23 kDa and 35 kDa proteins (~3-fold) but provided an opportunity for further enrichment through the 'clickable' ethynyl moiety. No proteins were specifically labeled by 32P-5N3-8-ethynyl-NAADP in sea urchin egg homogenate. These experiments demonstrate that 5N3-8-ethynyl-NAADP is biologically active and selectively labels putative NAADP-binding proteins in mammalian systems, evidencing a 'bifunctional' probe with utility for isolating NAADP-binding proteins.

A Bioactive Resveratrol Trimer from the Stem Bark of the Sri Lankan Endemic Plant Vateria copallifera.

A new resveratrol trimer, vateriferol (1), having four cis-oriented methine protons and constituting four contiguous stereocenters, was isolated from the bark extract of Vateria copallifera by bioassay-guided fractionation using a combination of normal, reversed phase, and size exclusion column chromatography. The structure was established based on its spectroscopic data. Vateriferol (1) was evaluated in vitro for its antioxidant capacity, enzyme inhibitory activity, growth inhibitory activity on a number of cancer cell lines, neuroprotective activity, and anti-inflammatory activity. Vateriferol (1) exhibited AChE inhibitory activity (IC50 8.4 ± 0.2 µM), ORAC activity (2079 ± 0.20 TE/g), and neuroprotective activity at 1.5 µM using PC12 cells deprived of oxygen and glucose and lowered NO levels in lipopolysaccharide-stimulated SIM-A9 microglial cells at 14.7 and 73.6 µM. Vateriferol (1) exhibited weak cytotoxic potency (<50% growth inhibition) against the tested cell lines at 147.2 µM.

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+) mobilizing second messenger discovered to date, has been implicated in Ca(2+) signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca(2+) signaling or the identity of the Ca(2+) stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca(2+) signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca(2+) signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca(2+) stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca(2+) signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca(2+) release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells.

Nicotinic Acid Adenine Dinucleotide Phosphate Analogues Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on ReceptorMediated Ca²âº Release.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+) releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20-500-fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca(2+) with an EC50 of 31 µM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced.

Activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs in a human cell line: difference in specificity between human and sea urchin NAADP receptors.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger that has been identified. We have previously shown that NAADP analogs substituted at the 5-position of nicotinic acid were recognized by the sea urchin receptor at low concentration, whereas the 4- substituted analogs were not as potent. However, to date the structure-activity relationship (SAR) of these analogs has not been addressed in mammalian systems. Thus, we asked whether these structurally modified analogs behave similarly in an NAADP-responsive mammalian cell line (SKBR3) using microinjection and single cell fluorescent imaging methods. Novel "caged" 4- and 5-substituted NAADP analogs that were activated inside the cell by flash photolysis resulted in Ca2+ mobilizing activity in SKBR3 cells in a concentration dependent manner, but with reduced effectiveness compared to unmodified NAADP. The SAR in mammalian SKBR3 cells was quite different from that of sea urchin and may suggest that there are differences between NAADP receptors in different species or tissues. Importantly, these data indicate that modifications at the 4- and 5-position of the nicotinic acid ring may lead to the development of functional photoaffinity labels that could be used for receptor localization and isolation in mammalian systems.

Nicotinic Acid Adenine Dinucleotide 2'-Phosphate (NAADP) Binding Proteins in T-Lymphocytes.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Although several channels, including two-pore channels (TPC), ryanodine receptor (RYR) and mucolipin (TRP-ML1) have been implicated in NAADP regulation of calcium signaling, the NAADP receptor has not been identified. In this study, the photoaffinity probe, [32P]-5-azido-NAADP ([32P]-5-N3-NAADP), was used to study NAADP binding proteins in extracts from NAADP responsive Jurkat T-lymphocytes. [32P]-5-N3-NAADP photolabeling of Jurkat S100 cytosolic fractions resulted in the labeling of at least ten distinct proteins. Several of these S100 proteins, including a doublet at 22/23 kDa and small protein at 15 kDa displayed selectivity for NAADP as the labeling was protected by inclusion of unlabeled NAADP, whereas the structurally similar NADP required much higher concentrations for protection. Interestingly, the labeling of several S100 proteins (60, 45, 33 and 28 kDa) was stimulated by low concentrations of unlabeled NAADP, but not by NADP. The effect of NAADP on the labeling of the 60 kDa protein was biphasic, peaking at 100 nM with a five-fold increase and displaying no change at 1 µM NAADP. Several proteins were also photolabeled when the P100 membrane fraction from Jurkat cells was examined. Similar to the results with S100, a 22/23 kDa doublet and a 15 kDa protein appeared to be selectively labeled. NAADP did not increase the labeling of any P100 proteins as it did in the S100 fraction. The photolabeled S100 and P100 proteins were successfully resolved by two-dimensional gel electrophoresis. [32P]-5-N3-NAADP photolabeling and two-dimensional electrophoresis should represent a suitable strategy in which to identify and characterize NAADP binding proteins.

Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.

Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.

Nicotinic acid adenine dinucleotide phosphate analogues containing substituted nicotinic acid: effect of modification on Ca(2+) release.

Analogues of nicotinic acid adenine dinucleotide phosphate (NAADP) with substitution at either the 4- or the 5-position position of the nicotinic acid moiety have been synthesized from NADP enzymatically using Aplysia californica ADP-ribosyl cyclase or mammalian NAD glycohydrolase. Substitution at the 4-position of the nicotinic acid resulted in the loss of agonist potency for release of Ca(2+)-ions from sea urchin egg homogenates and in potency for competition ligand binding assays using [(32)P]NAADP. In contrast, several 5-substituted NAADP derivatives showed high potency for binding and full agonist activity for Ca(2+) release. 5-Azido-NAADP was shown to release calcium from sea urchin egg homogenates at low concentration and to compete with [(32)P]NAADP in a competition ligand binding assay with an IC(50) of 18 nM, indicating that this compound might be a potential photoprobe useful for specific labeling and identification of the NAADP receptor.

Structural basis for nicotinamide inhibition and base exchange in Sir2 enzymes.

The Sir2 family of proteins consists of broadly conserved NAD(+)-dependent deacetylases that are implicated in diverse biological processes, including DNA regulation, metabolism, and longevity. Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base-exchange reaction to reform NAD(+) at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide bound to a Sir2 homolog, yeast Hst2, in complex with its acetyl-lysine 16 histone H4 substrate and a reaction intermediate analog, ADP-HPD. Together with related biochemical studies and structures, we identify a nicotinamide inhibition and base-exchange site that is distinct from the so-called "C pocket" binding site for the nicotinamide group of NAD(+). These results provide insights into the Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.

Carbocyclic ribosylamines: synthesis of 5-substituted carbocyclic beta-ribofuranosylamines.

A synthesis of 5-substituted cyclopentylamine precursors for 5'-substituted carbocyclic nucleoside analogues was developed. We show that the stereochemistry of the OsO4-catalyzed hydroxylation of an apically brominated lactam, 7-bromo-2-azabicyclo[2.2.1]hept-5-en-3-one, can be controlled through the appropriate selection of the lactam N-H protecting group. Sterically large groups direct the hydroxylation to the exo-face of the olefin, yielding hydroxylation products that can be converted into analogues of carbocyclic ribosides. Conversely, a sterically small protecting group permits OsO4 approach from the endo-face, yielding hydroxylation products analogous to carbocyclic lyxosides. A key intermediate for carbocyclic sugar production, (1S,2S,3R, 4R,5S)-1-(tert-butyloxycarbonyl)amino-5-bromo-2,3-(dimethylmethylene)dioxy-4-hydroxymethylcyclopentane, was synthesized starting from a commercially available enantiomerically pure lactam, (1S)-(+)-2-azabicyclo[2.2.1]hept-5-en-3-one, in seven steps in an overall yield of 21%.

Cyclic ADP-ribose analogues containing the methylenebisphosphonate linkage: effect of pyrophosphate modifications on Ca2+ release activity.

Analogues of cyclic ADP-ribose (cADPR) incorporating a methylenebisphosphonate linkage in the place of the pyrophosphate have been synthesized from nicotinamide adenine dinucleotide analogues enzymatically using Aplysia californica ADP-ribosyl cyclase. Methylenebisphosphonate cyclic ADP-ribose (cADPR[CH(2)]) and methylenebisphosphonate cyclic 3-deaza-ADP-ribose (3-deaza-cADPR[CH(2)]) showed full agonist activity for release of Ca(2+) ions from sea urchin egg homogenates. The EC(50) for cADPR[CH(2)] was 856 nM and that for 3-deaza-cADPR[CH(2)] was 300 nM, about 15- and 5-fold less potent than cADPR, respectively.

Substrate specificity and kinetic mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases.

The Silent information regulator 2 (Sir2) family of enzymes consists of NAD(+)-dependent histone/protein deacetylases that tightly couple the hydrolysis of NAD(+) and the deacetylation of an acetylated substrate to form nicotinamide, the deacetylated product, and the novel metabolite O-acetyl-ADP-ribose (OAADPR). In this paper, we analyzed the substrate specificity of the yeast Sir2 (ySir2), the yeast HST2, and the human SIRT2 homologues toward various monoacetylated histone H3 and H4 peptides, determined the basic kinetic mechanism, and resolved individual chemical steps of the Sir2 reaction. Using steady-state kinetic analysis, we have shown that ySir2, HST2, and SIRT2 exhibit varying catalytic efficiencies and display a preference among the monoacetylated peptide substrates. Bisubstrate kinetic analysis indicates that Sir2 enzymes follow a sequential mechanism, where both the acetylated substrate and NAD(+) must bind to form a ternary complex, prior to any catalytic step. Using rapid-kinetic analysis, we have shown that after ternary complex formation, nicotinamide cleavage occurs first, followed by the transfer of the acetyl group from the donor substrate to the ADP-ribose portion of NAD(+) to form OAADPr and the deacetylated product. Product and dead-end inhibition analyses revealed that nicotinamide is the first product released followed by random release of OAADPr and the deacetylated product.