6-Azido- and 6-aminoflavins as active-site probes of flavin enzymes.
6-Azidoflavins have been bound to the apoproteins of five representative flavoproteins and their properties, before and after light irradiation, compared with those of the same proteins containing the appropriate 6-aminoflavin. In the dark the 6-azidoflavoproteins are quite stable, except for L-lactate oxidase, where spontaneous conversion to the 6-amino-FMN enzyme occurs slowly at pH 7. 6-Azido-FMN Old Yellow Enzyme is converted to the 6-amino-FMN enzyme by aerobic turnover with NADPH, and 6-azido-FAD D-amino acid oxidase is converted to the 6-amino-FAD enzyme by treatment with D-alanine. Light irradiation of 6-azidoriboflavin bound to riboflavin-binding protein does not result in any covalent fixation of the flavin to the protein. Light irradiation of 6-azido-FMN flavodoxin gives only a small amount of covalent linkage. In contrast, 6-azido-FMN Old Yellow Enzyme undergoes a very facile light-induced change, in which approximately 50% of the flavin is attached in a stable covalent linkage to the protein. The resulting flavoprotein form has lost the ability to bind phenols, a distinctive characteristic of the native enzyme; it does, however, bind NADPH, but the latter cannot reduce the covalently bound flavin. 6-Azido-FAD D-amino acid oxidase also undergoes a facile light modification, in which almost quantitative fixation of the flavin to the protein takes place. The resulting flavoprotein cannot bind benzoate, an active-site ligand for the native enzyme, nor is it reduced anaerobically by D-alanine. The covalent linkage is fairly labile and is destroyed on denaturation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
D001386:Azides / Q000494:pharmacology*; D001665:Binding Sites; D003605:D-Amino-Acid Oxidase / Q000378:metabolism; D005415:Flavins / Q000494:pharmacology*; D005418:Flavodoxin / Q000378:metabolism; D005420:Flavoproteins / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000378:metabolism; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000494:pharmacology; D013053:Spectrophotometry
D001386:Azides; D005415:Flavins; D005418:Flavodoxin; D005420:Flavoproteins; C051370:6-azidoriboflavin; C051371:6-aminoriboflavin; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase; D003605:D-Amino-Acid Oxidase; D009252:NADPH Dehydrogenase; D012256:Riboflavin