L-aspartate oxidase from Escherichia coli. I. Characterization of coenzyme binding and product inhibition
Articolo
Data di Pubblicazione:
1996
Citazione:
L-aspartate oxidase from Escherichia coli. I. Characterization of coenzyme binding and product inhibition / M. Mortarino, A. Negri, G. Tedeschi, T. Simonic, S. Duga, H.G. Gassen, S. Ronchi. - In: EUROPEAN JOURNAL OF BIOCHEMISTRY. - ISSN 0014-2956. - 239:2(1996 Jul 15), pp. 418-426.
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 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.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
Binding; FAD; Inhibition; L-aspartate oxidase; Site-directed mutagenesis
Elenco autori:
M. Mortarino, A. Negri, G. Tedeschi, T. Simonic, S. Duga, H.G. Gassen, S. Ronchi
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