R. Jeck et al., NEW REACTIVE COENZYME ANALOGS FOR AFFINITY LABELING OF NAD(+) AND NADP(+) DEPENDENT DEHYDROGENASES, Zeitschrift fur Naturforschung. C, A journal of biosciences, 50(7-8), 1995, pp. 476-486
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.