Abstract
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 non-covalently 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 (K(d)) equal to 1.4 μM. The enzyme binds FAD by a simple second-order process with K(d) 0.67 μM. 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.
| Original language | English |
|---|---|
| Pages (from-to) | 418-426 |
| Number of pages | 9 |
| Journal | European Journal of Biochemistry |
| Volume | 239 |
| Issue number | 2 |
| Publication status | Published - 1996 |
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Keywords
- Binding
- FAD
- Inhibition
- L-aspartate oxidase
- Site-directed mutagenesis
ASJC Scopus subject areas
- Biochemistry
Cite this
L-Aspartate oxidase from Escherichia coli - I. Characterization of coenzyme binding and product inhibition. / Mortarino, Michele; Negri, Armando; Tedeschi, Gabriella; Simonic, Tatjana; Duga, Stefano; Gassen, Hans Gunther; Ronchi, Severino.
In: European Journal of Biochemistry, Vol. 239, No. 2, 1996, p. 418-426.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - L-Aspartate oxidase from Escherichia coli - I. Characterization of coenzyme binding and product inhibition
AU - Mortarino, Michele
AU - Negri, Armando
AU - Tedeschi, Gabriella
AU - Simonic, Tatjana
AU - Duga, Stefano
AU - Gassen, Hans Gunther
AU - Ronchi, Severino
PY - 1996
Y1 - 1996
N2 - 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 non-covalently 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 (K(d)) equal to 1.4 μM. The enzyme binds FAD by a simple second-order process with K(d) 0.67 μM. 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.
AB - 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 non-covalently 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 (K(d)) equal to 1.4 μM. The enzyme binds FAD by a simple second-order process with K(d) 0.67 μM. 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.
KW - Binding
KW - FAD
KW - Inhibition
KW - L-aspartate oxidase
KW - Site-directed mutagenesis
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UR - http://www.scopus.com/inward/citedby.url?scp=0030018052&partnerID=8YFLogxK
M3 - Article
VL - 239
SP - 418
EP - 426
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
SN - 0014-2956
IS - 2
ER -