Enzyme Function of the Globin Dehaloperoxidase from Amphitrite ornata Is Activated by Substrate Binding

Amphitrite ornata dehaloperoxidase (DHP) is a heme enzyme with a globin structure, which is capable of oxidizing para-halogenated phenols to the corresponding quinones. Cloning, high-level expression, and purification of recombinant DHP are described. Recombinant DHP was assayed by stopped-flow expe...

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Bibliographic Details
Published in:Biochemistry (Easton) 2005-12, Vol.44 (48), p.15637-15644
Main Authors: Belyea, Jennifer, Gilvey, Lauren B, Davis, Michael F, Godek, Marisha, Sit, Tim L, Lommel, Steven A, Franzen, Stefan
Format: Article
Language:English
Subjects:
Animals
Cloning, Molecular
Enzyme Activation
Escherichia coli - enzymology
Escherichia coli - genetics
Globins - metabolism
Hemoglobins
Hydrogen Peroxide - metabolism
Peroxidases - antagonists & inhibitors
Peroxidases - genetics
Peroxidases - metabolism
Phenols - metabolism
Polychaeta - enzymology
Protein Binding
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
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Summary:Amphitrite ornata dehaloperoxidase (DHP) is a heme enzyme with a globin structure, which is capable of oxidizing para-halogenated phenols to the corresponding quinones. Cloning, high-level expression, and purification of recombinant DHP are described. Recombinant DHP was assayed by stopped-flow experiments for its ability to oxidatively debrominate 2,4,6-tribromophenol (TBP). The enzymatic activity of the ferric form of recombinant DHP is intermediate between that of a typical peroxidase (horseradish peroxidase) and a typical globin (horse heart myoglobin). The present study shows that, unlike other known peroxidases, DHP activity requires the addition of substrate, TBP, prior to the cosubstrate, peroxide. The presence of a substrate-binding site in DHP is consistent with a two-electron oxidation mechanism and an obligatory order for activation of the enzyme by addition of the substrate prior to the cosubstrate.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi051731k