New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases
- 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.
Author: | Reinhard JeckGND, Michael Scholze, Anja Tischlich, Christoph Woenckhaus, Jürgen ZimmermannGND |
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URN: | urn:nbn:de:hebis:30:3-799385 |
DOI: | https://doi.org/10.1515/znc-1995-7-803 |
ISSN: | 0939-5075 |
ISSN: | 1865-7125 |
Parent Title (German): | Zeitschrift für Naturforschung, C |
Publisher: | Verlag der Zeitschrift für Naturforschung |
Place of publication: | Tübingen |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2014/06/02 |
Year of first Publication: | 1995 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2024/02/06 |
Tag: | Affinity Labeling; Dehydrogenases; NAD + Analogues; NADP+ Analogues |
Volume: | 50.1995 |
Issue: | 7-8 |
Page Number: | 11 |
First Page: | 476 |
Last Page: | 486 |
Institutes: | Medizin |
Biochemie, Chemie und Pharmazie / Biochemie und Chemie | |
Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Sammlungen: | Universitätspublikationen |
Licence (German): | Creative Commons - Namensnennung-Nicht kommerziell-Keine Bearbeitung 3.0 |