Preparation and Properties of 3-Halopyridine-adenine Dinucleotides, NAD + Analogues and of Model Compounds

Abdallah, Mohamed Abou-Elwafa; Biellmann, Jean‐François; Samama, Jean‐Pierre; Wrixon, A.D.

doi:10.1111/j.1432-1033.1976.tb10308.x

Cited by 27 publications

(16 citation statements)

References 30 publications

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“…A similar trend was Cinnamyl alcohol is a substrate of alcohol dehydrogenase in buffer with a Michaelis constant of 1.6 pM. It exhibits a substrate excess inhibition above 8 pM [34]. In microemulsions At-I and Ct with cinnamyl alcohol, a similar pattern is observed (Fig.…”

Section: Effect Of P H On the Michaelis Constants And Maximum Velocitysupporting

confidence: 65%

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Enzymes and microemulsions. Activity and kinetic properties of liver alcohol dehydrogenase in ionic water-in-oil microemulsions

1987

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The activity and the kinetic properties of horse liver alcohol dehydrogenase have been studied in water-in-oil microemulsions containing sodium dodecyl sulfate (SDS) or hexadecyl trimethylammonium bromide (CTAB), 1-butanol or I-pentanol or 1-hexanol or t-butanol, water and cyclohexane alone o r with octane. In the anionic microemulsions (i.e. containing sodium dodecyl sulfate), the enzyme quickly lost its activity, but was efficiently protected by the coenzyme and some adenine nucleotides. In the cationic microemulsions (i.e. containing hexadecyl trimethylammonium bromide), the enzyme activity was more stable and with higher alcohols was stable for at least 20 min. The Michaelis constant of NAD' calculated with respect to the water content was nearly constant and higher than in water. The maximum velocity in anionic microemulsions depends on the water content whereas in cationic microemulsions, the maximum velocity did not show a clear dependence on the water content and was close to the maximum velocity found in water. The pH dependence of K , and V,,, in these microemulsions was similar to that observed in water. The kinetic data for an hydrophobic substrate, cinnamyl alcohol, showed that this alcohol partitions between the pseudo-phases and thus the apparent Michaelis constant and the concentration at which substrate-excess inhibition appeared were increased. The catalytic properties of the enzyme in microemulsions were illustrated by the preparative reduction of cinnamaldehyde with cofactor recycling. The rate determination of NAD' reduction and of 1-butanol/cinnamaldehyde redox reaction showed that at low water content (2.8%), the NAD' reduction rate was close to zero whereas the redox reaction rate was about half of the rate at higher water content. Probably at low water content the coenzyme binding-dissociation rates are reduced much more than the binding-dissociation rates of the substrates and the rates of the ternary complex interconversion. The cationic microemulsions seemed to be a very favorable medium for enzyme activity, the tetraalkyl ammonium surfactant causing less denaturation than the anionic detergent dodecyl sulfate.The use of water-soluble enzymes for chemical synthesis suffers from several limitations. The most obvious are the low water solubility of many organic substances and the fact that the presence of water makes the reversal of some enzyme reactions for synthetic purposes difficult. In order to overcome these problems, much attention has been paid over the last few years to the solubilization of biological macromolecules in micellar solutions [I -51. Most of the published work deals with three component systems: water, organic solvent and surfactants. Water-soluble enzymes like ribonuclease, peroxidase, a-chymotrypsin, trypsin, lactate dehydrogenase, pyruvate kinase, pyrophosphatase, lysozyme and alcohol dehydrogenase have been solubilized [l -141 in swollen reverse micelles from water, octane or isooctane and di(2-ethylhexy1)sodium sulfosuccinate (AOT). In some instances, other su...

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Section: Effect Of P H On the Michaelis Constants And Maximum Velocitysupporting

confidence: 65%

“…The interesting and very useful point was the increase of the overall concentration of cinnamyl alcohol where substrate excess inhibition started to be detectable: 1 mM (Fig. 6) compared to 8 pM in water [34]. The cinnamaldehyde reduction with coenzyme recycling was then done in microemulsion according to Fig.…”

Section: Discussionmentioning

confidence: 99%

Enzymes and microemulsions. Activity and kinetic properties of liver alcohol dehydrogenase in ionic water-in-oil microemulsions

1987

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“…In contrast, the presence of an intact carbonyl oxygen atom is much more critical for catalytic activity in agreement with the X-ray observations. However, io3PdAD+ is an active analogue [2] in spite of the inactive mode of binding observed here. A possible explanation for this apparent contradiction would be that the complex exists in two different conformations in solution, one of which is active and the other having the observed inactive mode of binding.…”

Section: Discussionmentioning

confidence: 81%

“…As part of these studies, and also for thc use as heavy-atom derivatives in protein Crystallography, we synthesized 3-iOdO and subsequently 3-bromo and 3-chloro derivatives of pyridineadenine dinucleotide which were active in enzymatic hydrogen transfer reactions. Synthesis of these analogues and their kinetic properties for some NAD+-dependent dehydrogenase-catalysed reactions is presented elsewhere [2].…”

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confidence: 99%

The Crystal Structure of Complexes between Horse Liver Alcohol Dehydrogenase and the Coenzyme Analogues 3‐Iodopyridine‐adenine Dinucleotide and Pyridine‐adenine Dinucleotide

Samama

Zeppezauer

Biellmann

et al. 1977

European Journal of Biochemistry

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We have studied the binding of the enzymatically active NAD + analogue, 3-iodopyridine-adenine dinucleotide, and the inactive analogue, pyridine-adenine dinucleotide to the enzyme horse liver alcohol dehydrogenase using X-ray crystallographic methods. These studies were made under such conditions that crystals of the complexes were isomorphous to apocnzyme crystals. Both analogues bind in the same conformation. The binding of the adenosine moiety is very similar to that of ADP-ribose or NADH bound to the enzyme. The conformation and mode of binding of the remaining portions of the analogue molecules is, however, quite different. The pyridine ring is not situated in the active-site pocket as the nicotinamide group in thc isomorphous enzyme -NADH . imidazole complex but lies at the surface of the crevice between the two domains of the subunit, approximately 1.5 nm away from the catalytically active zinc atom. Lys-228 which has been shown to be important for NADH dissociation is in this region of the molecule.A large number of analogues to the coenzyme NAD' have been investigated [l] in order to delineate the role of the various moieties of the dinucleotide in the interaction of this coenzyme with the NAD+-dependent dehydrogenases. We have been particularly interested in the enzymatic effects of substitutions on the nicotinamide ring. As part of these studies, and also for thc use as heavy-atom derivatives in protein Crystallography, we synthesized 3-iOdO and subsequently 3-bromo and 3-chloro derivatives of pyridineadenine dinucleotide which were active in enzymatic hydrogen transfer reactions. Synthesis of these analogues and their kinetic properties for some NAD+-dependent dehydrogenase-catalysed reactions is presented elsewhere [2].The present study describes the binding of the 3-iodo analogue to one of these enzymes, horse liver alcohol dehydrogenase, as studied by X-ray crystallographic methods. Furthermore, the results of that Abbreviations.Pd AD + . pyridine-adenine dinucleot ide ; io3PdAD+, 3-iodopyridine-adenine dinucleotide; Tes, 2-([tris-(hydroxymethyl)methyi]amino)ethane sulfonic acid.

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“…The ADP-ribose molecule binds within the active site while PdADf is excluded from this area but still binds to the orthorhombic structure. Furthermore, the presence of some inhibitors (i. e. imidazole) can induce binding outside the active site of enzymatically active analogue like io3PdAD' [19].…”

Section: Discussionmentioning

confidence: 99%

5-Methylnicotinamide-adenine Dinucleotide. Kinetic Investigation with Major and Minor Isoenzymes of Liver Alcohol Dehydrogenase and Structural Determination of Its Binary Complex with Alcohol Dehydrogenase

1981

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5-Methylnicotinamide -adenine dinucleotide and 3-cyano-5-methylpyridine -adenine dinucleotide were prepared from 5-methylthionicotinamide -adenine dinucleotide by chemical conversion. The 5-methylthionicotinamide -adenine dinucleotide was obtained by enzymic transglucosidation. Model compounds ascertained the structure. None of the dinucleotides rnethylated at C-5 was active with the major isoenzyme EE of horse liver alcohol dehydrogenase, but activity with 5-methylnicotinaniide -adenine dinucleotide was measured with the minor isoenzymes. The binding of 5-methylnicotinamide -adenine dinucleotide to liver alcohol dehydrogenase, investigated by X-ray diffraction methods to 0.37-nm resolution, occurs with the pyridinium ring away from the active site as previously described for 3-iodopyridine -adenine and pyridine -adenine dinucleotides. A general conclusion on the use of inhibitors as tools for exploration of the active site is drawn.The X-ray structure determination of the orthorhombic apoenzyme of the alcohol dehydrogenase from horse liver [I] prompted the study of the binding of coenzyme or coenzyme analogues and substrates to clarify the binding mode and the reaction mechanism. It has been shown previously that when an oxidized or a reduced coenzyme is soaked into these crystals, they crack and dissolve. Crystallisation of ternary eiizyme-NADH-substrate complexes gives triclinic or monoclinic crystals with a different conformation of the enzyme molecule [2]. During a screening of coenzyme analogues it was found that 5-methylnicotinamide-adenine dinucleotide (m5NAD)+ did not induce cracking of the orthorhombic crystals in spite of earlier reports that this analogue showed activity as an hydride acceptor [3]. This apparent discrepancy between the activity and absence of crystal cracking prompted us to carry out a more thorough chemical and biochemical study of this analogue and of model compounds. We report here the results of our chemical and biochemical investigations and the structure determination at 0.37-nm resolution of the orthorhombic binary complex alcohol dehydrogenase-m5NAD+. A study of m5NAD+ with lactate dehydrogenase has been reported recently [4]. Enzymes. Alcohol dehydrogenase (EC 1.1.1.1); lactate dehydrogenase (EC 1 .I .1.27); NAD(P)+ glycohydrolase (EC 3.2.2.6); glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). MATERIALS AND METHODS 5- MODEL COMPOUNDS3-Cyano-5-methylpyridine (1). A suspension of 5-methylnicotinamide (3 g) in phosphorous oxychloride ( 5 ml) was heated under reflux for 45 min. After cooling, the excess of reagent was removed under reduced pressure and the mixture poured into a concentrated sodium carbonate solution and extracted thrice with chloroform. The extract was dried over sodium sulfate. Removal of the solvent under reduced pressure gave a solid which was sublimed (65°C under 1.33 Pa). The 3-cyano-5-methyl pyridine (I) was obtained as white crystals (1.93 g, yield 74%): m.p. 82-84°C (cf. 83-84°C [6]). ' H NMR (C'HCI,): 6 = 2.43 ppm (3H), 7.81 ppm (IH) and 8.69 p...

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Preparation and Properties of 3-Halopyridine-adenine Dinucleotides, NAD + Analogues and of Model Compounds

Cited by 27 publications

References 30 publications

Enzymes and microemulsions. Activity and kinetic properties of liver alcohol dehydrogenase in ionic water-in-oil microemulsions

Enzymes and microemulsions. Activity and kinetic properties of liver alcohol dehydrogenase in ionic water-in-oil microemulsions

The Crystal Structure of Complexes between Horse Liver Alcohol Dehydrogenase and the Coenzyme Analogues 3‐Iodopyridine‐adenine Dinucleotide and Pyridine‐adenine Dinucleotide

5-Methylnicotinamide-adenine Dinucleotide. Kinetic Investigation with Major and Minor Isoenzymes of Liver Alcohol Dehydrogenase and Structural Determination of Its Binary Complex with Alcohol Dehydrogenase

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