New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases
| Autor: | Jürgen Zimmermann, Reinhard Jeck, Michael Scholze, Anja Tischlich, Christoph Woenckhaus |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Affinity labeling
Magnetic Resonance Spectroscopy biology Nicotinamide Chemistry Affinity label Dehydrogenase Affinity Labels NAD Medicinal chemistry General Biochemistry Genetics and Molecular Biology Cofactor Substrate Specificity Adenosine diphosphate chemistry.chemical_compound Kinetics Structure-Activity Relationship biology.protein Animals Indicators and Reagents NADH NADPH Oxidoreductases NAD+ kinase Chromatography High Pressure Liquid NADP Alcohol dehydrogenase |
| Zdroj: | Zeitschrift fur Naturforschung. C, Journal of biosciences. 50(7-8) |
| ISSN: | 0939-5075 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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