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Title
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Position 713 is critical for catalysis but not iron binding in soybean lipoxygenase 3.
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Authors
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J.A.Kramer,
K.R.Johnson,
W.R.Dunham,
R.H.Sands,
M.O.Funk.
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Ref.
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Biochemistry, 1994,
33,
15017-15022.
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PubMed id
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Abstract
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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.
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