Position 713 Is Critical for Catalysis but Not Iron Binding in Soybean Lipoxygenase 3

Jeffrey A. Kramer, Keith R. Johnson, William R. Dunham, Richard H. Sands, Max O. Funk

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

The role of asparagine-713 in iron atom incorporation and catalysis in soybean lipoxygenase 3 was investigated using site-directed mutagenesis. A full-length cDNA for the lipoxygenase isoenzyme was obtained from a library derived from soybeans cv. Provar. Protein with native specific activity at pH 7.4 was obtained from expression in Escherichia coli. Two recent structure reports provided conflicting views about the participation of the side chain of asparagine-694 in the coordination of the iron atom required for catalysis by lipoxygenase 1. Oligonucleotide-directed mutagenesis was employed to modify residue 713 in lipoxygenase 3 which corresponds to asparagine-694 in the sequence of lipoxygenase 1. It was found that for enzyme expressed in bacteria, asparagine was not required for iron incorporation. Histidine, alanine, and serine substitutions for asparagine-713 all produced proteins that contained iron. The histidine mutant had specific activity and catalytic characteristics comparable to the wild-type enzyme. By contrast, the alanine- and serine-substituted lipoxygenases had no detectable catalytic activity. When oxidized by product, the histidine mutant also displayed the characteristic g6 signal of the soybean enzyme in its EPR spectrum. The possibilities that the residue at position 713 acts as a metal ligand, an acid-base catalyst, and a hydrogen bonding group are considered and discussed.

Original languageEnglish (US)
Pages (from-to)15017-15022
Number of pages6
JournalBiochemistry
Volume33
Issue number50
DOIs
StatePublished - Dec 1 1994

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Asparagine
Catalysis
Soybeans
Iron
Lipoxygenase
Histidine
Mutagenesis
Site-Directed Mutagenesis
Alanine
Serine
Enzymes
Lipoxygenases
Atoms
Hydrogen Bonding
Oligonucleotides
Escherichia coli
Isoenzymes
Libraries
Paramagnetic resonance
Byproducts

ASJC Scopus subject areas

  • Biochemistry

Cite this

Position 713 Is Critical for Catalysis but Not Iron Binding in Soybean Lipoxygenase 3. / Kramer, Jeffrey A.; Johnson, Keith R.; Dunham, William R.; Sands, Richard H.; Funk, Max O.

In: Biochemistry, Vol. 33, No. 50, 01.12.1994, p. 15017-15022.

Research output: Contribution to journalArticle

Kramer, Jeffrey A. ; Johnson, Keith R. ; Dunham, William R. ; Sands, Richard H. ; Funk, Max O. / Position 713 Is Critical for Catalysis but Not Iron Binding in Soybean Lipoxygenase 3. In: Biochemistry. 1994 ; Vol. 33, No. 50. pp. 15017-15022.
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