Spectroscopic studies of pyridoxamine (pyridoxine) 5'-phosphate oxidase. Equilibrium dissociation constants and spectra for riboflavin 5'-phosphate and analogues.
Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been shown to bind 1 mol of riboflavin 5'-phosphate (FMN) per mol of apoenzyme and is active with or inhibited by numerous FMN analogues [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436--3442]. The KD values and spectra for selected apoenzyme--flavin complexes have been determined and used to elucidate some of the properties of the FMN-binding site of this flavoprotein. Alterations of the pyrimidinoid portion of the flavin ring decrease binding considerably. The absorption spectra for the protein complexes with 3-deaza-FMN and 8-hydroxy-FMN indicate the presence of a dipolar or positively charged protein group near N1 and O2. The substitution of methyl for hydrogen at N3 apparently causes distortion of the interaction between the flavin ring and an active-site aromatic amino acid residue. Although binding is also decreased somewhat by substitutions at postions 8 and 8 alpha, considerable bulk [e.g., 8-(diethylamino)-FMN and 8 alpha-S-(N-acetyl-cysteinyl)-FMN] is accommodated. Hence, this portion of the flavin ring is probably oriented toward, possibly in contact with, solvent, as has been found for the flavodoxins. The importance of optimum interactions between the flavin and the apoprotein is further emphasized by large differences in the activity of flavin analogues that have similar midpoint potentials in solution.
D001051:Apoenzymes; D001665:Binding Sites; D002942:Circular Dichroism; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives*; D007700:Kinetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011734:Pyridoxaminephosphate Oxidase / Q000378:metabolism*; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D001051:Apoenzymes; D005486:Flavin Mononucleotide; D011734:Pyridoxaminephosphate Oxidase; D000587:Oxidoreductases Acting on CH-NH Group Donors