New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases.
Reactive coenzyme analogues omega-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of omega-(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 to 0 degree C. Lactate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazoniumpentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3 alpha, 20 beta-Hydroxysteroid dehydrogenase reacts at 0 degree C with the ethyl homologue and slowly with the propyl compound. The butyl- and pentyl analogues do not inactivate at 0 degree 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 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 dehydrogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP+-dependent dehydrogenases the compound has been shown 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.
Jeck
R
R
Klinikum der Johann Wolfgang Goethe-Universität, Bundesrepublik Deutschland.
Scholze
M
M
Tischlich
A
A
Woenckhaus
C
C
Zimmermann
J
J
D000345:Affinity Labels / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000345:Affinity Labels; D007202:Indicators and Reagents; D009243:NAD; D009249:NADP; D009247:NADH, NADPH Oxidoreductases