D-aspartate oxidase from beef kidney. Purification and properties.
The flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been purified to homogeneity from beef kidney cortex. The protein is a monomer with a molecular weight of 39,000 containing 1 molecule of flavin. The enzyme as isolated is a mixture of a major active form containing FAD and a minor inactive form containing 6-hydroxy-flavin adenine dinucleotide (6-OH-FAD). The absorption and fluorescence spectral properties of the two forms have been studied separately after reconstitution of the apoprotein with FAD or 6-OH-FAD, respectively. FAD-reconstituted D-aspartate oxidase has flavin fluorescence, shows characteristic spectral perturbation upon binding of the competitive inhibitor tartaric acid, is promptly reduced by D-aspartic acid under anaerobiosis, reacts with sulfite to form a reversible covalent adduct, stabilizes the red anionic form of the flavin semiquinone upon photoreduction, and yields the 3,4-dihydro-FAD-form after reduction with borohydride. A Kd of 5 X 10(-8) M was calculated for the binding of FAD to the apoprotein. 6-OH-FAD-reconstituted D-aspartate oxidase has no flavin fluorescence, shows no spectral perturbation in the presence of tartaric acid, is not reduced by D-aspartic acid under anaerobiosis, does not stabilize any semiquinone upon photoreduction, and does not yield the 3,4-dihydro-form of the coenzyme when reduced with borohydride; the enzyme stabilizes the p-quinoid anionic form of 6-OH-FAD and lowers its pKa more than two pH units below the value observed for the free flavin. The general properties of the enzyme thus resemble those of the dehydrogenase/oxidase class of flavoprotein, particularly those of the amino acid oxidases.
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D050804:D-Aspartate Oxidase; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008433:Mathematics; D010777:Photochemistry; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D005182:Flavin-Adenine Dinucleotide; C052277:6-hydroxy-FAD; D000594:Amino Acid Oxidoreductases; D050804:D-Aspartate Oxidase