Reinhard Jeck

Modification of alcohol dehydrogenases with two NAD+-analogues containing reactive substituents on the functional side of the molecule

A coenzyme analogue with a reactive bromoacetyl group on the non-functional side of the molecule has been used previously to identify different cysteine residues at the active sites of alcohol dehydrogenases [ 1 ] . These cysteine residues are known to be ligands to the zinc atom at the catalytic centre [2]. Analogues with reactive substituents corresponding to other positions on the coenzyme molecule are potentially of interest for mapping the active sites of this and other dehydrogenases. In the present work, therefore, labelled residues in yeast and horse liver alcohol dehydrogenases have been identified after modifications with two other NAD analogues, 14C-labelled [3-(3-bromoacetylpyridinio)propyl] -adenosine pyrophosphate and [3-(4-bromoacetylpyridinio)-propyl] -adenosine pyrophosphate. Both analogues inactivated the yeast enzyme under conditions suggesting binding at the coenzyme-site [3,4], and led to covalent incorporation of 1 molecule of analogue per protein subunit [5,6]. The horse enzyme reacted similarly with the 4-bromoacetylpyridinio derivative but was not inactivated by the other analogue. Labelled residues in the proteins were identified by analysis of radioactive peptides after digestion with chymotrypsin. A limited variability in the labelling with different analogues was established, showing that the active sites of both alcohol dehydrogenases contain few reactive residues, in agreement with data from crystallographic studies of the horse enzyme [2] and results of structural comparisons [7].

Simple methods of preparing nicotinamide mononucleotide

Nicotinamide mononucleotide (NMN) is the functional moiety of the coenzyme NAD ~, starting material for the preparation of many coenzyme analogs with an altered nonfunctional moiety [1]. NMN is obtained by enzymatic cleavage of the NAD~-pyrophosphate bond using NAD~-pyrophospha tase (dinucleotide-nucleotido hydrolase, EC 3.6.1.9) [2,3]. Moreover, several chemical methods of synthesizing the compound have been described [4-7]. This paper is to report the preparation of NMN starting with NAD~-employing NAD~-pyrophospha tase from potatoes bound to a supporting matrix as well as a simple chemical method using 2,3-O-isopropylidine-D-ribofuranosylamine as starting material [8].

A simple preparation of an enzyme reactor producing nicotinamidemononucleotide

SummaryAn enzyme reactor which produces nicotinamidemononucleotide is easily prepared by adsorption of NAD pyrophosphatase to phosphocellulose. The separation and purification of the mononucleotide is achieved in a single chromatographic step. The spectrophotometric data of purified NMN and its cyanide adduct were redetermined.

[24] Alcohol dehydrogenases

Publisher Summary NAD is the coenzyme of alcohol dehydrogenase. It serves in the reversible enzymic reaction as an acceptor of the hydrogen from the substrate and is converted to NADH. This occurs in a ternary complex of enzyme, pyridine nucleotide, and substrate, in which coenzyme and substrate interact closely with amino acid residues of the enzyme, and thereby are activated sufficiently to allow reaction. This chapter describes the synthesis of acetyl pyridinio-n-alkyl monophosphates, and synthesis of dinueleotide analogs. It also explains the preparation of the dihydrocoenzyme analogs, preparation of inactivating reagents, and assay of inhibitor, preparation of the covalently linked enzyme-coenzyme compounds, kinetics of inactivation, optical properties of enzyme-coenzyme analog compounds, identification of amino acid residues, oxidation of the inactivator-enzyme compound, and stabilization of the bond between coenzyme and protein.

Photoaffinity labelling of lactate dehydrogenase from pig heart with a bifunctional NAD(+)-analogue.

P1-N6-(4-azidophenylethyl)adenosine-P2-4-(3-azidopyridinio)b utyl diphosphate was synthesized with an [8-14C]adenine label. This bifunctional photoaffinity labelling reagent inactivates lactate dehydrogenase from pig heart upon irradiation with light of wavelength 300-380 nm. Stoichiometry of binding and enzymatic parameters suggest that the analogue is bound to the coenzyme binding site and that adjacent residues are modified. Four radioactive peptides were isolated by reverse-phase HPLC after tryptic digestion of the labelled protein. Amino-acid sequence analysis identified the peptides and correlation with the three-dimensional structure of dogfish lactate dehydrogenase reveals that the peptides correspond to positions affecting the coenzyme binding site, consistent with proper affinity labelling. Two of the peptides, Ile-77 --> Lys-81 and Asp-82 --> Asn-88, are located close to the adenine binding site. Low recovery of Thr-86 in combination with the detection of additional products in the sequence analysis indicates that this residue is modified by the photoaffinity label. The two other peptides (positions 119-124 and 318-328) are located next to the substrate binding site; their label is lost upon treatment with pyrophosphatase, showing that they are linked to the pyridinio moiety of the coenzyme analogue.

[9] Simple methods for preparing nicotinamide mononucleotide and related analogs

Publisher Summary This chapter describes the methods for preparing nicotinamide mononucleotide and related analogs. Nicotinamide mononucleotide (NMN) is a prerequisite in the preparation of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) analogs, of which the nonfunctional nucleotide moiety is to be modified; correspondingly, NMN analogs are necessary for preparing coenzyme models that have a modified functional moiety. There are two ways to prepare NMN: (1) cleavage of the dinucleotide NAD(P), by acid hydrolysis or by dinucleotide-nucleotidohydrolases (NAD-pyrophosphatases) and (2) total synthesis, starting with the nucleotide components nicotinamide, ribose, and phosphate. Nicotinamide nucleosides can be prepared from l-halogeno sugars and nicotinamide, or by the reaction of N 1 -(2,4-dinitrophenyl)-3-carba-moylpyridinium chloride with 1-amino sugars. In both cases, a mixture of anomeric nucleosides is obtained; the ratio of its constituents depends on the sugar compound. A selective phosphorylation of pyridinium nucleosides is very difficult. Either the introduction of protective groups or their removal afterward can pose problems. α-l-chloro-2,3,4-tri,- O -acetylglucopyranose has been used as the sugar compound to prepare the NMN analog nicotinamide-N l -β- D -glucopyranosyl-6-phosphate. The free hydroxy group in the 6-position of this sugar allows the selective phosphorylation later on and the acetyl group in the cis -position to the halogen favors the formation of the β-anomer.

Halbseiten-Reaktivität der Glycerinaldehyd-3-phosphat Dehydrogenase aus Kaninchen-Skelettmuskel mit Strukturanalogen von NAD / Half-of-the-Sites Reactivity of Glyceraldehyde-3 Phosphate Dehydrogenase from Rabbit Muscle with Structural Analogs of NAD

Abstract Alkylating NAD-Analogs, Glyceraldehyde-3 Phosphate Dehydrogenase, Half-of-the-Sites Reactivity co-(3-Bromoacetylpyridinio)alkyldiphosphoadenosines with alkyl chain lengths of 2 -6 me thylene groups inactivate glyceraldehyde-3 phosphate dehydrogenase from rabbit muscle. Half-of-the-Sites reactivity is observed in each case: The analogs are covalently bound to highly reactive cysteine residues in two of the four subunits. The remaining two subunits still bind N AD and the reactive SH-groups, although modified by SH-reagents of low molecular weight are not labeled by any of the brominated coenzyme models. This behaviour may be explained by the assumption, that the modification of 2 subunits induces structural changes in the neighboured unoccupied subunits which prevent any attack on reactive cysteine residues caused by fixation and orientation of the bromoketo-coenzyme analog when bound to the active center. Structural similarities of the covalently bound coenzyme analogs in the active center and the native ternary GAPDH-NAD-substrate complex suggest that half-of-the-sites reactivity is a natural characteristic of the enzymes catalytic mechanism.

Interaction between dehydrogenases and a new NAD -isomer.

Abstract A new NAD⊕-isomer was prepared, in which the ᴅ-ribose of the adenosine moiety was sub stituted by the enantiomeric ʟ-ribose. As compared to nicotinamide-adenine-dinucleotide (NAD⊕) and NADH the coenzyme isomer (ᴅ,ʟ)-NAD⊕ and its dihydroform (ᴅ,ʟ)-NADH are far less tightly bound to lactate dehydrogenase and alcohol dehydrogenase from horse liver. In the presence of the second substrate (ᴅ,ʟ)-NAD⊕ and (ᴅ,ʟ)-NADH act as hydrogen acceptor and hydrogen donator, respectively, with lactate dehydrogenase and alcohol dehydrogenases from horse liver and yeast. Compared to NAD⊕ and NADH the Michaelis constants are always increased, the catalytic constants (V/Et) were found to be decreased except for the dihydroform reacting with alcohol dehydrogenase from liver.

New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases.

Abstract Reactive coenzyme analogues ω-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of ω-(3-aminopyridinium)alkyl adenosine diphosphate with nitrous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability at 0 °C. L actate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazonium pentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3α,20β-Hydroxysteroid dehydrogenase reacts at 0 °C with the ethyl homologue and slowly with the propyl compound. The butyl-and pentyl analogues do not inactivate at 0 °C. Tests with 14C -labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)butyl 2 ′-phospho-adenosine diphosphate, a structural analogue of NADP +, was prepared by condensation of adenosine-2,3-cyclophospho-5′-phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid ester with 2 ′:3′-cyclonucleotide-3′-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehyd rogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP +-dependent dehydrogenases the com pound has been show n to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase.

Phosphohydrolase Activities in Developing and Mature Dental Tissues

Abstract In the course of the odontogenesis of bovine incisors several clearly distinguishable phosphohydrolase activities are observed in the pulp and in dental hard tissues. Using various substrates and inhibitors, unspecific alkaline phosphatase, two isoenzymes of acid phosphatase, Ca2+-activated ATPase and inorganic pyrophosphatase are characterized. The enzymatic activity of alkaline phosphatase in pulp and hard tissues is significantly high at the beginning of dentine and enamel mineralization. The specific activity of this enzyme decreases quite fast with the beginning of root formation, then more slowly, until it reaches a constant final value. Histochemical studies show that during mineralization the maximum of alkaline phosphatase activity is in the subodontoblasts. Lower enzyme concentrations are found in the stratum intermedium and in the outer enamel epithelium during that process. The specific activities of ATPase, acid phosphatases and pyrophosphatase show little temporal variation during tooth development, but they also appear in a characteristic spatial pattern in the dental tissues.