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The properties of the NAD-binding domain of Bungarus fasciatus venom NAD glycohydrolase were investigated by a number of biochemical techniques. Inhibitor studies indicated that an intact dinucleotide was required for effective enzyme recognition. Adenosine derivatives, AMP, ADP, ATP, adenosine diphosphoribose (ADP-rib), and 2'-phosphoadenosine diphosphoribose (phospho-ADP-rib), were linear competitive inhibitors of the NADase-catalyzed reaction. Nicotinamide analogs were noncompetitive inhibitors with Ki values that varied with the nature and position of substituent groups. Selective enzyme inactivation by 2,4-pentanedione indicated the importance of a lysinyl residue for NADase activity. The importance of a carboxyl group was also indicated by the sensitivity of the NADase toward N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K). Reagents which modified sulfhydryl, histidyl, and arginyl residues were not effective in inactivating B. fasciatus venom NADase. The purified snake venom NADase catalyzed a transglycosidation (pyridine base exchange) reaction. The functioning of a variety of substituted pyridine bases in this reaction was demonstrated by the formation of the corresponding NAD analogs. Kinetic studies of the transglycosidation reaction were consistent with the partitioning of an enzyme-ADP-rib intermediate between water and the substituted pyridine base. Concentrations of pyridine bases required for enzyme inhibition differed significantly from those needed for the pyridine base exchange reaction, suggesting two sites or two forms of the NADase differing in affinity for pyridine bases. A number of NAD analogs were also shown to be effective ribosyl donors in the transglycosidation reaction.

Reactions catalyzed by purified Bungarus fasciatus venom NAD glycohydrolase were demonstrated to include ADP-ribose transfer from NAD to alcohols and to imidazole derivatives to produce a variety of ADP-ribosides. The formation of products was monitored by high performance liquid chromatography. In the enzyme-catalyzed alcoholysis of NAD, the ratio of n-alkyl-ADP-riboside formed to the hydrolytic product, ADP-ribose, increased linearly with alcohol concentration. The effectiveness of alcohols as acceptors of the ADP-ribose moiety in these reactions increased with increasing chainlength of the alcohol used. Linear positive chainlength effects extended from methanol to pentanol suggesting facilitation of these reactions by nonpolar interactions. In the methanolysis reaction, NADP, thionicotinamide adenine dinucleotide, nicotinamide-1, N6-ethenoadenine dinucleotide, and 3-acetylpyridine adenine dinucleotide were shown to be as effective as NAD as donor substrates. The NAD glycohydrolase-catalyzed ADP-ribose transfer to pyridine bases to form NAD analogs was studied at pyridine base concentrations above those determined to be saturating for the base exchange reaction. Under these conditions, the ratio of base exchange to hydrolysis of NAD was directly related to the pKa of the ring nitrogen of the pyridine base employed. In addition to alcoholysis and pyridine-base exchange reactions, the snake venom enzyme was demonstrated to catalyze an ADP-ribose transfer reaction to imidazole derivatives. Arginine methyl ester was ineffective as an ADP-ribose acceptor molecule in these reactions.

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.