Iron extraction from soybean lipoxygenase 3 and reconstitution of catalytic activity from the apoenzyme.
Lipoxygenases contain a unique nonheme iron cofactor with a redox role in the catalyzed reaction. The conditions for the extraction of the metal atom were investigated for one of the soybean lipoxygenase isoenzymes. Removal of the iron by o-phenanthroline was attained in the presence of substrate under anaerobic conditions, but the apoenzyme could not be isolated and reconstituted. The freshly regenerated sodium form of Chelex-100 also removes the iron atom from native soybean lipoxygenase 3, but only in sodium bicarbonate buffer at pH 8.0. The soluble but inactive apoenzyme was reconstituted with ferric ammonium sulfate in Tris--HCl buffer at pH 7.0. Stoichiometric iron in the reconstituted enzyme was established using inductively coupled plasma-atomic emission spectroscopy. The reconstituted enzyme contained 90 +/- 10% of the specific activity of the native enzyme. The native configuration of the reconstituted iron site was confirmed by electron paramagnetic resonance spectroscopy.
Kariapper
M S
MS
Department of Chemistry, University of Toledo, Toledo, Ohio 43606, USA.
Dunham
W R
WR
Funk
M O
MO
D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002614:Chelating Agents / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry*; D007502:Iron Chelating Agents / Q000737:chemistry; D008084:Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D010618:Phenanthrolines / Q000737:chemistry; D012117:Resins, Synthetic; D013025:Glycine max / Q000201:enzymology*
D001051:Apoenzymes; D002614:Chelating Agents; D007502:Iron Chelating Agents; D008667:Metalloproteins; D010618:Phenanthrolines; D012117:Resins, Synthetic; C024997:Chelex 100; D007501:Iron; C095864:lipoxygenase 3; D008084:Lipoxygenase; C025205:1,10-phenanthroline