D-aspartate oxidase from beef kidney. Purification and properties

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• D-aspartate oxidase (EC 1.4.3.1) is a flavoprotein found in beef kidney cortex.
• The enzyme plays a role in amino acid metabolism, similar to other amino acid oxidases.

Purpose of the Study:

• To purify and characterize D-aspartate oxidase from beef kidney.
• To investigate the biochemical and spectral properties of the enzyme's active (FAD-bound) and inactive (6-OH-FAD-bound) forms.

Main Methods:

• Homogeneous purification of D-aspartate oxidase.
• Spectroscopic analysis (absorption and fluorescence) of reconstituted enzyme forms.
• Enzyme kinetics and cofactor binding studies.

Main Results:

• Purified D-aspartate oxidase is a monomer (39,000 MW) containing one flavin molecule.
• The active form utilizes FAD, exhibiting fluorescence and characteristic spectral changes with D-aspartic acid and tartaric acid.
• The inactive form contains 6-hydroxy-flavin adenine dinucleotide (6-OH-FAD), lacks fluorescence, and shows altered reactivity and spectral properties.

Conclusions:

• D-aspartate oxidase exists as FAD and 6-OH-FAD containing forms with distinct biochemical properties.
• The FAD-bound form exhibits typical flavoprotein oxidase characteristics, including substrate and inhibitor interactions.
• The 6-OH-FAD form demonstrates altered cofactor binding and reduced enzymatic activity.