8-Azidoacenine analogs of NAD+ and FAD. Synthesis and coenzyme properties with NAD+-dependent and FAD-dependent enzymes.
The synthesis and purification of the 8-azidoadenine analogs of NAD+ (azido-NAD+) and FAD (AZIDO-FAD) from 8-azidoadenosine 5'-phosphate and NMN+ or FMN, respectively, is described. The coenzyme analogs are characterized by absorption, nuclear magnetic resonance and circular dichroism spectra. The two latter methods indicate a folded structure of azido-NAD+ and azido-FAD. Upon irradiation at 300 mn in aqueous solution, a change of the ultraviolet absorption spectra of the coenzyme analogs indicates photolysis of the azido group. The coenzyme properties of azido-NAD+ are demonstrated with lactate, glutamate and alcohol dehydrogenase yielding 14, 154 and 60%, respectively, of the V observed with NAD+. Concomitantly, the Km values of the coenzyme analogs are 1.7, 3.5 and 3-fold higher than those of NAD+. Azido-FAD is shown to be coenzyme of apo-glucose oxidase. The recovery of activity, however, is much slower in the presence of azido-FAD than with FAD. A final value of 66% of the activity with FAD is obtained. With apo-D-amino acid oxidase, azido-FAD is completely inactive, although it is specifically bound to the enzyme.
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D002942:Circular Dichroism; D003605:D-Amino-Acid Oxidase / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives*; D005949:Glucose Oxidase / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009243:NAD / Q000031:analogs & derivatives*; D010782:Photolysis; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D001386:Azides; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D000429:Alcohol Oxidoreductases; D007770:L-Lactate Dehydrogenase; D005949:Glucose Oxidase; D005969:Glutamate Dehydrogenase; D003605:D-Amino-Acid Oxidase