Inhibition studies of dehydrogenases by structural analogues of NAD.

The coenzyme anologues [3-(2-acetylpyridinio) propyl]-adenosine pyrophosphate and [3-(2-bromoacetylpyridinio) -propyl] -adenosine pyrophoshate were synthesized and used for inhibition studies. While the first compound reacts as competitive inhi­ bitor against NAD+, the latter is able to form co­ valently bound derivatives of several NAD+-dependent dehydrogenases, giving informations about amino acid side chains participating in the binding of the functional coenzyme part. [3(2-acetylpyridinio) -propyl] -adenosine pyro­ phosphate exhibits strong hypochromicity between adenine and 2-acetylpyridine ring. Cleavage of the pyrophosphate bridge increases the absorption at 261 nm up to 22% and indicates, that the stacked configuration of the molecule is preferred in aqueous solution. In enzymatic assays with YADH and GAPDH [3(2-acetylpyridinio) -propyl] -adenosine pyrophos­ phate acts as a competitive inhibitor against NAD+. The inhibition constants are: Xi = 1.7'10-2 M (YADH) and 4.3,10“4m (GAPDH) and show, that the coenzyme analogue has little affinity to the co­ enzyme binding sites. Difference spectra of the coenzyme-enzyme mixtures and the isolated com­ ponents do not show any spectral changes due to the formation of the binary complex in the case of YADH and only small effects with GAPDH. From this results it can be concluded, that the incorpora­ tion of the 2-acetylpyridinio-propyl residue into the enzymes is sterically hindered. This is also indicated by the fact, that YADH shows smaller inhibition constants with adenosine monophosphate and adeno­ sine diphosphate 1. We obtained [3(2-bromoacetylpyridinio) -pro­ pyl]-adenosine pyrophosphate by bromination of the acetyl group of the coenzyme analogue. The brominated compound is unstable in aqueous solu­ tion: At pH 6.5 the halftime is 120 min. At pH 7.5 the time is 20 min and at pH 8.5 it is 3 min.