L-aspartate oxidase from Escherichia coli. I. Characterization of coenzyme binding and product inhibition.
This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of noncovalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant (Kd) equal to 1.4 microM. The enzyme binds FAD by a simple second-order process with Kd 0.67 microM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.
Mortarino
M
M
Istituto di Fisiologia Veterinaria e Biochimica, Università di Milano, Italy.
Negri
A
A
Tedeschi
G
G
Simonic
T
T
Duga
S
S
Gassen
H G
HG
Ronchi
S
S
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D029968:Escherichia coli Proteins; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D000594:Amino Acid Oxidoreductases; C031955:L-aspartate oxidase, E coli