Calf-spleen nicotinamide-adenine dinucleotide glycohydrolase. Properties of the active site.

The interaction between the nicotinamide adenine dinucleotide binding domain of calf spleen NAD glycohydrolase and its ligands has been studied. The use of competitive inhibitors, structurally related to different portions of the NAD molecule (i.e. adenosine and nicotinamide moieties), revealed the considerable importance of the binding between the pyrophosphate linkage and probably an arginyl residue of the active site. This interaction allows the positioning of the substrate in a conformation which permits catalysis to occur. The binding between the 2'-hydroxyl of the adenosine moiety and a residue of the active site, which exists in NAD-linked dehydrogenases, is probably missing in the calf spleen NAD glycohydrolase, based on the inhibition by salicylates, 2'-deoxyadenosine 5'-monophosphate and the hydrolysis of the 2'-deoxyadenosine analogue of NAD. The NAD glycohydrolase could be completely inactivated by 2,3-butanedione, an arginyl-modifying reagent. The reaction followed pseudo-first-order kinetics and the modification was found to be reversible. Woodward's reagent K, a reagent for carboxyl residues, partially inactivated the enzyme, which resulted in a change of the NAD glycohydrolase kinetic parameters Km and V. The inactivation rate was complicated by a parallel decomposition of the reagent.     

Calf-spleen nicotinamide--adenine dinucleotide glycohydrolase. Solubilization purification and properties of the enzyme.

NAD glycohydrolase of calf spleen was solubilized with pancreatic lipase and purified approximatively 800-fold to a specific activity of 7 units/mg of protein by successive DEAE-cellulose and carboxymethyl-cellulose chromatography. The purified enzyme has a molecular weight of 24,000 and is characterized by a double band on disc gel electrophoresis. Some kinetic properties of the NAD-glycohydrolase-catalyzed hydrolsis of NAD have been examined using a titrimetric assay for enzyme activity. The reaction is subject to inhibition be excess of substrate, which disappears at high ionic strength and low pH. At a pH below 5 the kinetic displays an apparent activation by substrate. The effects of pH (4.5-9.0) on the kinetic parameters do not reveal an essential ionizable group in the catalytic process.     

NAD+ Glycohydrolase of the Plasma Membrane Prepared from Glial and Neuronal Cells

NAD+ glycohydrolase (EC 3.2.2.5) activity was detected in the plasma membrane prepared from the primary culture of rat astrocytes. The enzyme has a broad optimum pH range. From the kinetic analysis, a Michaelis constant of 91.2 microM and a maximum velocity of 0.785 mumol/min/mg protein were obtained. ADPribose exhibited a competitive inhibition with respect to NAD. The inhibition by nicotinamide was shown to be of a non-competitive type. ATP and GTP were found to be competitive inhibitors. NAD+ glycohydrolase activity was not detected in the plasma membrane prepared from the primary culture of neuronal cells of chick embryos.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. IX. Nicotinamide adenine dinucleotide glycohydrolase: a plasma membrane enzyme prominently found in Kupffer cells

The distribution of nicotinamide adenine dinucleotide (NAD) glycohydrolase in rat liver was investigated by subcellular fractionation and by isolation of hepatocytes and sinusoidal cells. The behavior of NAD glycohydrolase in subcellular fractionation was peculiar because, although the enzyme was mainly microsomal, plasma membrane preparations contained distinctly more NAD glycohydrolase than could be accounted for by their content in elements derived from the endoplasmic reticulum or the Golgi complex identified by glucose-6-phosphatase and galactosyltransferase, respectively. When microsomal and plasmalemmal preparations were brought to equilibrium in a linear-density gradient, NAD glycohydrolase differed from these enzymes and behaved like 5'-nucleotidase and alkaline phosphodiesterase I. NAD glycohydrolase was markedly displaced towards higher densities after treatment with digitonin. This behavior in density-gradient centrifugation strongly suggests that NAD glycohydrolase is an exclusive enzyme of the plasma membrane. NAD glycohydrolase differed clearly from other plasmalemmal enzymes when the liver was fractionated into hepatocytes and sinusoidal cells; its specific activity was considerably greater in sinusoidal cell than in hepatocyte preparations. Further subfractionation of sinusoidal cell preparations into endothelial and Kupffer cells by counterflow elutriation showed that NAD glycohydrolase is more active in Kupffer cells. We estimate that the specific activity of NAD glycohydrolase activity is at least 65-fold higher at the periphery of Kupffer cells than at the periphery of hepatocytes. As the enzyme shows not structure-linked latency and is an exclusive constituent of the plasma membranes, we conclude that it is an ectoenzyme that cannot lead to a rapid turnover of the cytosolic pyridine nucleotides.     

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.     

Thyroid membrane ADP ribosyltransferase activity. Stimulation by thyrotropin and activity in functioning and nonfunctioning rat thyroid cells in culture

Bovine thyroid membranes possess both ADP ribosyltransferase and NAD glycohydrolase activities with the same Km values for NAD and the same pH optima. In intact membranes, the ADP ribosyltransferase is limited in its extent by the amount of available membrane acceptor which can be ADP-ribosylated; in membranes solubilized with lithium diiodosalicylate, an artificial acceptor, L-arginine methyl ester, can be substituted to eliminate this limitation. The product of the ADP ribosyltransferase is a mono-ADP-ribosylated acceptor whether the intact or solubilized membrane provides the enzyme activity and whether membrane or exogenous acceptor, L-arginine methyl ester, is utilized. The intact membranes and the solubilized preparation also have an enzyme activity which can release AMP from the mono-ADP-ribosylated acceptor whether formed by the action of the membrane ADP ribosyltransferase or the A promoter of cholera toxin. The NAD glycohydrolase activity appears to represent the half-reaction of the ADP ribosyltransferase, i.e. an activity measurable substituting water for a membrane acceptor or L-arginine methyl ester. Membranes from functional rat thyroid cells in culture, i.e. cells chronically stimulated by thyrotropin and unresponsive to further additions of thyrotropin, have low ADP-ribosylation but high NAD glycohydrolase activities. In contrast, membranes from nonfunctional rat thyroid cells, i.e. cells unresponsive to thyrotropin, have high ADP-ribosylation and low NAD glycohydrolase activities. NAD hydrolysis by the NAD glycohydrolase activity cannot account for the low ADP-ribosylation activity in membranes from the functioning cells, and its low level of ADP-ribosylation can be eliminated by solubilizing the membranes and substituting an artificial acceptor, L-arginine methyl ester. The ADP ribosyltransferase activity of rat thyroid cell membrane preparations can be enhanced by thyrotropin in a dose-dependent manner but not by insulin, glucagon, hydrocortisone, adrenocorticotropin, or its glycoprotein hormone analog, human chorionic gonadotropin. It is thus suggested (i) that, in analogy to cholera toxin, thyrotropin-stimulated ADP-ribosylation may be important in the regulation of the adenylate cyclase response and (ii) that the level of membrane acceptor available for ADP-ribosylation may relate both to a stable "'activated" state of the adenylate cyclase system in cells chronically stimulated with thyrotropin and/or to a desensitized state with regard to a failure of more thyrotropin to elicit additional functional responses.     

Asymmetric reassociation of calf spleen NAD+ glycohydrolase into liposomes.

NAD+ glycohydrolase (NAD+ nucleosidase, EC 3.2.2.6) can be solubilized from calf spleen microsomes (microsomal fractions) by steapsin or by detergents to yield respectively a hydrophilic (i.e. water-soluble) and a hydrophobic form of the enzyme. The detergent-solubilized enzyme was successfully reassociated into phosphatidylcholine liposomes either by a cholate-dialysis or by a gel-filtration procedure. In both cases the incorporation of NAD+ glycohydrolase was found to be completely asymmetric, i.e. the active site of the enzyme was exposed only at the outer surface of the vesicles. By contrast, as judged by flotation experiments, the hydrophilic form of NAD+ glycohydrolase could not be reassociated into liposomes. These results are in agreement with the hypothesis that calf spleen NAD+ glycohydrolase is an amphipathic protein. When incorporated into large unilamellar vesicles composed of phosphatidylcholine, NAD+ glycohydrolase was not found to catalyse vectorial transfer of NAD+ by transglycosidation with nicotinamide as acceptor.     

NAD Glycohydrolases: A possible function in calcium homeostasis

NAD glycohydrolases are the longest known enzymes that catalyze ADP-ribose transfer. The function of these ubiquitous, membrane-bound enzymes has been a long standing puzzle. The NAD glycohydrolase are briefly reviewed in light of the discovery by our laboratory that NAD glycohydrolases are bifunctional enzymes that can catalyze both the synthesis and hydrolysis of cyclic ADP-ribose, a putative second messenger of calcium homeostasis.Abbreviations NADase nicotinamide adenine dinucleotide glycohydrolase - NAD nicotinamide adenine dinucleotide - ADP-ribose adenosine diphosphoribose - cADPR cyclic adenosine diphosphoribose     

Studies on the association of NAD glycohydrolase with membranes in calf spleen

The subcellular distribution of NAD glycohydrolase was studied by fractionation of calf spleen homogenates using differential and discontinuous density gradient centrifugations. The highest amount of NAD glycohydrolase activity was associated with microsomes, which in this tissue were found to contain, in addition to endoplasmic reticulum, a large proportion of vesicles derived from plasma membranes. The distribution pattern of NAD glycohydrolase was found to parallel that of plasma membrane markers. When microsomal vesicles were treated with digitonin, NAD glycohydrolase activity and plasma membranes specifically increased in density. We conclude that in calf spleen the bulk of NAD glycohydrolase is associated with plasma membranes. Microsomal NAD glycohydrolase was associated with sealed vesicles; its activity could not be increased by disruption of the sidedness of the vesicles. This result and further observations based on the known restricted permeability of biological membranes to charged substances, and on the activity of the enzyme with non-penetrating substrates and inhibitors, indicate that the NAD glycohydrolase active site is located on the exterior side of the vesicles. It is proposed that calf spleen NAD glycohydrolase is an ecto-enzyme.     

Localization of oxidized nocotinamide--adenine dinucleotide glycohydrolase in the mouse liver nuclear envelope.

NAD+ glycohydrolase activity located in the nuclear envelope was maximally solubilized by treatment with 0.1--0.2% Triton X-100. The residual activity largely represents the chromatin-associated NAD+ glycohydrolase. Under these conditions the phospholipids were extensively solubilized (over 90%) while leaving the nuclei physically stable, although the nuclear membranes were removed, as shown by electron microscopy. After Triton X-100 treatment, deoxyribonuclease I did not significantly affect the residual NAD+ glycohydrolase activity, although the DNA was completely broken down. This enzyme activity can be released from the nuclear pellet by incubation with phospholipase C. For comparative studies, the glucose 6-phosphatase activity, known to be present in the nuclear envelope, was investigated. Treatment with 0.01% Triton X-100 released 10--20% of the phospholipids, but without solubilizing either glucose 6-phosphatase or NAD+ glycohydrolase. Higher Triton X-100 concentrations (0.1--1.0%) inhibited glucose 6-phosphatase, but not NAD+ glycohydrolase activity. NAD+ glycohydrolase is apparently present in a latent form in the nuclear envelope. Glucose 6-phosphatase, However, shows no such latency.     

Slow-binding inhibition of NAD+ glycohydrolase by arabino analogues of beta-NAD.

Modifications at the 2'-position of the nicotinamide-ribosyl moiety influence dramatically the nature of the interactions of the modified beta-NAD+ with calf spleen NAD+ glycohydrolase (EC 3.2.2.6), an enzyme that cleaves the nicotinamide-ribose bound in NAD(P)+. Nicotinamide arabinoside adenine dinucleotide (ara-NAD+) and nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide (araF-NAD+) are not hydrolyzed at measurable rates and are the first documented examples of reversible slow binding inhibitors of this class of enzyme. The kinetic data obtained are consistent with both slow kon and koff rate constants in the formation of an enzyme-inhibitor complex, i.e. the association rate constants are about 10(4) and 10(6) slower than diffusion rates, respectively, for araF-NAD+ and ara-NAD+, and the half-life of the complex is about 3-10 min for both analogues. The kinetic model does not account for a slow turnover of an ADP-ribosyl-enzyme intermediary complex. AraF-NAD+ is one of the most potent inhibitors described for NAD+ glycohydrolase.     

Metabolism of nicotinamide mononucleotide in beef liver

Nicotinamide mononucleotide (NMN) is not only an intermediate for the biosynthesis but also a degradation product of pyridine cofactors in animal tissues. Among the animal tissues tested, the highest NMN catabolizing activity was detected in beef liver (5.6 mumol/min/g tissue). This activity was 16 times higher than the NAD hydrolysis catalyzed by the liver NAD glycohydrolase. As a result of enzymatic analysis of the NMN splitting process, two types of enzyme responsible for this catabolism were partially purified and identified as a membrane-bound 5'-nucleotidase and a cytoplasmic nicotinamide riboside (NR) phosphorylase. No specific NMN glycohydrolase could be found in contrast to results observed in bacterial systems. The 5'-nucleotidase and NR phosphorylase constitute an obligatory process of the pyridine nucleotide cycle. The dephosphorylation and phosphorolysis catalyzed suggest that these enzymes could serve as an important mechanism for salvaging the ribose and nicotinamide moieties of NMN and pyridine nucleotides in the cell and a process that could be regulated at the mononucleotide level by this "NMN cycle" rather than by a NAD glycohydrolase cycle. In addition to the enzymatic properties of these enzymes, a regulatory mechanism by nucleotides such as ATP was also demonstrated.     

NAD Glycohydrolase Activities and ADP-Ribose Uptake in Erythrocytes From Normal Subjects and Cancer Patients

Erythrocytes from cancer patients exhibited up to fivefold higher NAD glycohydrolase activities than control erythrocytes from normal subjects and also similarly increased [14C] ADP-ribose uptake values. When [adenosine-14C] NAD was used instead of free [14C] ADP-ribose, the uptake was dependent on ecto-NAD glycohydrolase activity. This was reflected in the inhibition of ADP-ribose uptake from [adenosine-14C] NAD by Cibacron Blue. ADP-ribose uptake in erythrocytes appeared to be complex: upon incubation with free [14C] ADP-ribose, the radiolabel associated with erythrocytes was located in nearly equal parts in cytoplasm and plasma membrane. Part of [14C] ADP-ribose binding to the membrane was covalent, as indicated by its resistance to trichloroacetic acid-treatment. A preincubation with unlabeled ADP-ribose depressed subsequent erythrocyte NAD glycohydrolase activity and binding of [14C] ADP-ribose to erythrocyte membrane; but it failed to inhibit the transfer of labeled ADP-ribose to erythrocyte cytoplasm. On the other hand, incubation with [adenosine-14C] NAD did not result in a similar covalent binding of radiolabel to erythrocyte membrane. In line with this finding, a preincubation with unlabeled NAD was not inhibitory on subsequent NAD glycohydrolase reaction and ADP-ribose binding. ADP-ribose binding and NAD glycohydrolase activities were found also in solubilized erythrocyte membrane proteins and, after size fractionation, mainly in a protein fraction of around 45kDa-molecular weight.     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of the soluble NAD glycohydrolase from Bungarus fasciatus venom

The NAD glycohydrolase (NADase) (EC 3.2.2.5) from Bungarus fasciatus (banded krait) venom was purified (1000-fold) to electrophoretic homogeneity through a 3-step purification procedure, the last step being affinity chromatography on Cibacron blue agarose. The purified NADase is a glycoprotein containing two subunits of Mr = 62,000 each. Nicotinamide and adenosine diphosphoribose were produced in a 1:1 stoichiometry and were the only products formed when the purified NADase was incubated with NAD. These results were confirmed by high performance liquid chromatography. The enzyme exhibited a brod pH profile with optimum pH for hydrolysis at 7.5 with very little change in Km from pH 6.0 to pH 8.5. The NADase is only slightly affected by changes in ionic strength. The enzyme studied titrimetrically at pH 7.5 and 38 degrees C exhibited a Km of 14 microM and a Vmax of 1380 mumol of NAD cleaved/min/mg of protein. The activation energy for the enzyme-catalyzed hydrolysis of NAD was 15.7 kcal/mol. In addition to NAD and NADP, a number of NAD analogs were shown to function as substrates for the enzyme. Product inhibition studies demonstrated nicotinamide to be a noncompetitive inhibitor with a KI of 1.5 mM and adenosine diphosphoribose a competitive inhibitor with a KI of 0.36 mM. Procion blue HB (Cibacron blue F3GA) was shown to be a competitive inhibitor with a KI of 33 nmol. The purified NADase catalyzed the pyridine base exchange reaction between 3-acetylpyridine and the nicotinamide moiety of NAD.     

Influence of phospholipids on the activity of phosphate-dependent glutaminase in extracts of rat liver mitochondria.

Liver glutaminase can be solubilized from frozen-and-thawed mitochondria by treatment with phospholipase A2. Solubilization by this technique markedly changes the kinetic properties of the enzyme. The properties of the membrane-bound form of the enzyme are partially restored by adding phosphatidylcholine or phosphatidylethanolamine to the phospholipase extract. It is concluded that the kinetic properties of liver glutaminase are a function of the interaction of this enzyme with membrane phospholipids.     

Topography, purification and characterization of thyroidal NAD+ glycohydrolase

Subcellular fractionation of bovine thyroid tissue by differential pelleting and isopycnic gradient centrifugation in a zonal rotor indicated that NAD(+) glycohydrolase is predominantly located and rather uniformly distributed in the plasma membrane. Comparison of NAD(+) glycohydrolase activities of intact thyroid tissue slices, functional rat thyroid cells in culture (FRT(l)) and their respective homogenates indicated that most if not all of the enzyme (catalytic site) is accessible to extracellular NAD(+). The reaction product nicotinamide was predominantly recovered from the extracellular medium. The diazonium salt of sulphanilic acid, not penetrating into intact cells, was able to decrease the activity of intact thyroid tissue slices to the same extent as in the homogenate. Under the same conditions this reagent almost completely abolished NAD(+) glycohydrolase activity associated with intact thyroid cells in culture. The triazine dye Cibacron Blue F3GA and its high-M(r) derivative Blue Dextran respectively completely eliminated or caused a severe depression in the NAD(+) glycohydrolase activity of FRT(l) cells. The enzyme could be readily solubilized from bovine thyroid membranes by detergent extraction, and was further purified by gel filtration and affinity chromatography on Blue Sepharose CL-6B. The overall procedure resulted in a 1940-fold purification (specific activity 77.6mumol of nicotinamide released/h per mg). The purified enzyme displays a K(m) of 0.40mm for beta-NAD(+), a broad pH optimum around pH7.2 (0.1 m-potassium phosphate buffer) and an apparent M(r) of 120000. Nicotinamide is an inhibitor (K(i) 1.9mm) of the non-competitive type. The second reaction product ADP-ribose acts as a competitive inhibitor (K(i) 2.7mm). The purified enzyme splits beta-NAD(+), beta-NADP(+), beta-NADH and alpha-NAD(+) at rates in the relative proportions 1:0.75:<0.02:<0.02 and exhibits transglycosidase (pyridine-base exchange) activity. Anionic phospholipids such as phosphatidylinositol and phosphatidylserine inhibit the partially purified enzyme. A stimulating effect was observed upon the addition of histones.     

Adenine nucleotides promote dissociation of pertussis toxin subunits

Pertussis toxin is composed of an enzymatically active A subunit and a binding component (B oligomer). Both the holotoxin and the isolated A subunit have previously been shown to exhibit NAD glycohydrolase activity although the A subunit is more active on a molar basis than the holotoxin. We have investigated the mechanism by which ATP stimulates the activity of this toxin. Since dissociation of pertussis toxin subunits would result in increased NAD glycohydrolase activity, the ability of ATP to promote release of the A subunit from the B oligomer was examined. In the presence of the zwitterionic detergent 3-(3-cholamidopropyldimethyl)-1-ammonio)-propanesulfonate, concentrations of ATP as low as 1 microM promoted subunit dissociation. The concentration of ATP required for release of the A subunit was similar to that required for stimulation of NAD glycohydrolase activity. Both ATP and ADP promoted subunit dissociation and stimulated NAD glycohydrolase activity. In contrast, AMP and adenosine did not alter NAD glycohydrolase activity or affect subunit structure. The ability of ATP to decrease the affinity of the A subunit for the B oligomer may play a role in nucleotide stimulation of pertussis toxin activity.     

Characterization of the NAD+ glycohydrolase associated with the rat liver nuclear envelope.

The localization of NAD+ glycohydrolase [EC 3.2.2.5] (NADase) in purified rat liver nuclei has been examined. Subnuclear fractionation revealed that at least 70% of the NADase in nuclei was associated with the nuclear envelope fraction. The nuclear envelope fraction was practically free of microsomal contamination as judged by electron microscopic morphometry and assays of microsomal marker enzymes. Therefore, NADase was found to be an integral component of the nuclear envelope. The enzymological properties of the nuclear envelope NADase were compared with those of the microsomal enzyme. The nuclear envelope NADase was identical to the microsomal enzyme in its Km for NAD+ (60 muM), pH optimum (pH 6.5), ratio of transglycosidase activity to NADase activity (about 0.5), thermal stability and sensitivity to various inhibitors. Thus, NADase is a common enzymic component of both the nuclear envelope and the endoplasmic reticulum.     

Nicotinamide 2-fluoroadenine dinucleotide unmasks the NAD+ glycohydrolase activity of Aplysia californica adenosine 5'-diphosphate ribosyl cyclase

ADP-ribosyl cyclases catalyze the transformation of nicotinamide adenine dinucleotide (NAD+) into the calcium-mobilizing nucleotide second messenger cyclic adenosine diphosphoribose (cADP-ribose) by adenine N1-cyclization onto the C-1' ' position of NAD+. The invertebrate Aplysia californica ADP-ribosyl cyclase is unusual among this family of enzymes by acting exclusively as a cyclase, whereas the other members, such as CD38 and CD157, also act as NAD+ glycohydrolases, following a partitioning kinetic mechanism. To explore the intramolecular cyclization reaction, the novel nicotinamide 2-fluoroadenine dinucleotide (2-fluoro-NAD+) was designed as a sterically very close analogue to the natural substrate NAD+, with only an electronic perturbation at the critical N1 position of the adenine base designed to impede the cyclization reaction. 2-Fluoro-NAD+ was synthesized in high yield via Lewis acid catalyzed activation of the phosphoromorpholidate derivative of 2-fluoroadenosine 5'-monophosphate and coupling with nicotinamide 5'-monophosphate. With 2-fluoro-NAD+ as substrate, A. californica ADP-ribosyl cyclase exhibited exclusively a NAD+ glycohydrolase activity, catalyzing its hydrolytic transformation into 2-fluoro-ADP-ribose, albeit at a rate ca. 100-fold slower than for the cyclization of NAD+ and also, in the presence of methanol, into its methanolysis product beta-1' '-O-methyl 2-fluoro-ADP-ribose with a preference for methanolysis over hydrolysis of ca. 100:1. CD38 likely converted 2-fluoro-NAD+ exclusively into the same product. We conclude that A. californica ADP-ribosyl cyclase can indeed be classified as a multifunctional enzyme that also exhibits a classical NAD+ glycohydrolase function. This alternative pathway that remains, however, kinetically cryptic when using NAD+ as substrate can be unmasked with a dinucleotide analogue whose conversion into the cyclic derivative is blocked. 2-Fluoro-NAD+ is therefore a useful molecular tool allowing dissection of the kinetic scheme for this enzyme.