The hydrolysis of NAD+ analogs bearing different substituents in the pyridinium moiety, catalyzed by solubilized calf spleen NAD+ glycohydrolase, was studied. The enzyme was specific for analogs possessing a carbonyl function at position C-3. The observed maximal rates showed no simple dependence either on the leaving ability of the parent pyridines or on the observed binding energies. The analogs without carbonyl substituents were not found to be hydrolyzed under our experimental conditions; and, compared to the substrates, they all presented much higher affinities for the active site, which could not be related to specific interactions. These results indicate that the rate-limiting step of the NAD+ hydrolysis, which is the formation of an enzyme-ADP-ribosyl intermediate, is probably more complex than a simple chemical step, i.e., pyridinium-ribose bond breakage. At a molecular level we favor a catalytic mechanism which, through nonbonded interactions between the substrate and the active site of the enzyme, results in the destabilization of the pyridinium-ribose bond, i.e., unimolecular decomposition involving an oxocarbonium ion intermediate.

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.

FEBS LettersVolume 73, Issue 1 p. 92-96 Full-length articleFree Access ADP-ribonolactone: A potential activated intermediate analogue of NAD-glycohydrolase First published: January 01, 1977 https://doi.org/10.1016/0014-5793(77)80023-9Citations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume73, Issue1January 01, 1977Pages 92-96 ReferencesRelatedInformation

Bioseparation is an important process in making biological products with a high degree of purity. The development of separation and purification strategies for bioproducts from diluted aqueous solutions has been identified as a critical need for obtaining specialty bioproducts and industrial chemicals. These diluted aqueous solutions are from fermentation, plant cell culture, or whole plant materials. Affinity chromatography has been an effective method for purification of bioproducts because of its simplicity and high degree of specificity. Affinity purification is carried out in columns packed with porous beads to which the affinity purification is carried out in columns packed with porous beads to which the affinity ligand is immobilized. The target biomolecule binds to the immobilized ligand via a specific interaction and then is eluted from the affinity column in high purity. However, the compressibility, slow mass transfer, and adsorption kinetics of the traditional particle media for column chromatography have significantly limited their application in purification of bioproducts on a large scale.

FEBS LettersVolume 66, Issue 1 p. 107-109 Full-length articleFree Access The stereochemistry of calf spleen NAD-glycohydrolase-catalyzed NAD methanolysis Marc Pascal, Marc Pascal Laboratoire de Physiologie Végétale, Institut de Botanique de l'Université Louis Pasteur, 28, rue Goethe, 67000 Strasbourg, FranceSearch for more papers by this authorFrancis Schuber, Corresponding Author Francis Schuber Laboratoire de Physiologie Végétale, Institut de Botanique de l'Université Louis Pasteur, 28, rue Goethe, 67000 Strasbourg, FranceTo whom correspondence should be addressedSearch for more papers by this author Marc Pascal, Marc Pascal Laboratoire de Physiologie Végétale, Institut de Botanique de l'Université Louis Pasteur, 28, rue Goethe, 67000 Strasbourg, FranceSearch for more papers by this authorFrancis Schuber, Corresponding Author Francis Schuber Laboratoire de Physiologie Végétale, Institut de Botanique de l'Université Louis Pasteur, 28, rue Goethe, 67000 Strasbourg, FranceTo whom correspondence should be addressedSearch for more papers by this author First published: July 01, 1976 https://doi.org/10.1016/0014-5793(76)80596-0Citations: 36AboutReferencesRelatedInformationPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessClose modalShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1 S.P. Colowick, Eys J. Van, J.H. Park, M. Florkin E.H. Stotz Comprehensive Biochemistry 14, (1966), Elsevier Publishing Co Amsterdam 1– 98. 2 N.O. Kaplan, M.M. Ciotti, F.E. Stolzenbach, J. Biol. Chem., 221, (1956), 833– 844. 3 B.M. Anderson, N.O. Kaplan, J. Biol. Chem., 234, (1959), 1226– 1232. 4 S.G.A. Alivisatos, Nature, 183, (1959), 1034– 1037. 5 F. Schuber, P. Aravo, Eur. J. Biochem., (1976), in the press 6 F. Schuber, P. Travo, M. Pascal, 10th International Congress FEBS (Paris), (1975), Abstract 608 7 T. Imoto, L.N. Johnson, A.C.T. Worth, D.C. Phillips, J.A. Rupley, P.D. Boyer 3rd Edn. The Enzymes 7, (1972), Academic Press New York 665– 868. 8 R. Barker, H.G. Fletcher Jr., J. Org. Chem., 26, (1961), 4605– 4609. 9 I.I. Secemski, S.S. Lehrer, G.E. Lienhard, J. Biol. Chem., 247, (1972), 4740– 4748. 10 R.L. Switzer, P.D. Simcox, J. Biol. Chem., 249, (1974), 5304– 5307. 11 M.L. Sinnott, O.M. Viratelle, Biochem. J., 133, (1973), 81– 87. Citing Literature Volume66, Issue1July 01, 1976Pages 107-109 ReferencesRelatedInformation Metrics Citations: 36 Details FEBS Letters 66 (1976) 1873-3468 © 2015 Federation of European Biochemical Societies Publication History Issue Online: 19 October 2001 Version of Record online: 19 October 2001 Manuscript received: 14 April 1976

Steady‐state analysis of product inhibition indicates that the most probable minimum kinetic mechanism for soluble calf spleen NAD glycohydrolase is ping‐pong bi‐bi, reducing to an ordered uni‐bi for hydrolysis alone. In the hydrolysis of NAD nicotinamide is released first. Isotope exchange, transglycosidation reaction and nucleophilic competition with methanol show that the formation of the intermediary enzyme · adenosine‐diphosphoribosyl complex (E‐ADP‐Rib) is rate limiting.The NAD hydrolysis is subject to inhibition by excess of substrate, which is markedly accentuated by increasing concentrations of nicotinamide. Kinetic analysis of this behaviour is in favour of the formation of an apparent dead‐end complex of NAD with the Michaelis complex.The reactivies of methanol and nicotinamide compared to water with the intermediate E‐ADP‐Rib suggest the occurrence of an intermediary oxocarbonium ion in the reaction catalyzed by NAD glycohydrolase.