Limited proteolysis and active-site labeling studies of soybean lipoxygenase.
Soybean lipoxygenase 1 was studied using limited proteolysis and active-site labeling to begin the structural characterization of the enzyme in solution. The serine proteases trypsin and chymotrypsin cleaved the large monomeric protein (95 kDa) into two large polypeptides, a C-terminal fragment of about 30 kDa and an N-terminal fragment of about 60 kDa. Under conditions that led to complete cleavage of the protein as judged by SDS-polyacrylamide gel electrophoresis, the catalytic activity of the protein was either reduced slightly (chymotrypsin) or enhanced (trypsin). The characteristics of the cleaved enzymes were the same as for native lipoxygenase 1 in all aspects examined: insensitivity to cyanide, fluoride, and EDTA, regiochemical and stereochemical consequences of catalysis, and EPR spectroscopy upon oxidation by product. The two fragments apparently were tightly associated as they could not be resolved under conditions which preserved the catalytic activity. Both native and protease-cleaved lipoxygenase 1 formed covalent adducts when treated with either 14C-phenylhydrazine or 4-nitrophenylhydrazine. The label was found only in the 60-kDa fragment and following complete trypsin digestion was associated with a peptide beginning after Lys-482 in the primary sequence. Therefore labeling occurred in the vicinity of the conserved histidine cluster which has been postulated as the iron-binding site. From these observations it appears that lipoxygenase 1 exists as a pair of tightly associated domains with the iron-binding site located in the larger of the two.
Ramachandran
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Department of Chemistry, University of Toledo, Ohio 43606.
Carroll
R T
RT
Dunham
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WR
Funk
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MO
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D008084:Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D011489:Protein Denaturation; D013025:Glycine max / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D008084:Lipoxygenase; D002918:Chymotrypsin; D014357:Trypsin