Position 713 is critical for catalysis but not iron binding in soybean lipoxygenase 3.
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.
Kramer
J A
JA
Department of Chemistry, University of Toledo, Ohio 43606.
Johnson
K R
KR
Dunham
W R
WR
Sands
R H
RH
Funk
M O
MO
D001216:Asparagine; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007501:Iron / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D011994:Recombinant Proteins; D001216:Asparagine; D007501:Iron; D008084:Lipoxygenase; D015246:Deoxyribonuclease EcoRI; C037106:endodeoxyribonuclease NdeI; D015252:Deoxyribonucleases, Type II Site-Specific