NEW REACTIVE COENZYME ANALOGS FOR AFFINITY LABELING OF NAD(+) AND NADP(+) DEPENDENT DEHYDROGENASES

Reactive coenzyme analogues omega-(3-diazoniumpyridinium)alkyl adenosi ne diphosphate were prepared by reaction of omega-(3-aminopyridinium)a lkyl adenosine diphosphate with nitrous acid. in these compounds the n icotinamide ribose is substituted by hydrocarbon chains of varied leng ths (n-ethyl to n-pentyl). The diazonium compounds are very unstable a nd decompose rapidly at room temperature. They show a better stability at 0 degrees C. Lactate and alcohol dehydrogenase do not react with a ny of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts r apidly 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 degrees C with the e thyl homologue and slowly with the propyl compound. The butyl- and pen tyl analogues do not inactivate at 0 degrees C. Tests with C-14-labele d 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that compl ete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)bu tyl 2'-phospho-adenosine diphosphate, a structural analogue of NADP(+) , was prepared by condensation of adenosine-2,3-cyclophospho-5'-phosph omorphoric date with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid ester with 2':3'-cyclonucleot ide-3'-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analog ue is a hydrogen acceptor and its reduced form a hydrogen donor in tes ts with alcohol dehydrogenase from Thermoanaerobium brockii. The reduc ed form of the coenzyme analogue also is a hydrogen donor with glutath ione reductase. With other NADP(+)-dependent dehydrogenases the compou nd has been shown to be a competitive inhibitor against the natural co enzyme. The acetyl group reacts with bromine to form the bromoacetyl g roup. This reactive bromoacetyl analogue is a specific active-site dir ected irreversible inhibitor of isocitrate dehydrogenase.