Lipoxygenases from soybeans and rabbit reticulocytes: inactivation and iron release.
Various inactivation methods were applied to lipoxygenases from soybean (isoenzyme 1) and rabbit reticulocytes to compare the inactivation behaviour of both enzymes and to elucidate the state of the iron which is known to be involved in the catalytic reaction of lipoxygenases: 1. Titration of the enzyme with mercury compounds shows that there are one or two SH groups responsible for the loss of activity in the presence of mercury. The SH groups seem not to be involved in the tight iron binding. 2. Inactivation by chelating agents such as o-phenanthroline or batho-phenanthroline sulfonic acid occurs only in the presence of reducing agents (mercaptoethanol and ascorbic acid). Our data support a co-oxidation mechanism. The complexation of iron by chelators is not the rate-limiting step. Both lipoxygenases show a very similar behaviour in this respect despite the fact that the reticulocyte enzyme requires the addition of trace amounts of copper ions for efficient inactivation. 3. Release of iron from the enzyme is also achieved by denaturation with guanidinium hydrochloride (Gu-HCl). In all cases, inactivation and release of iron were irreversible processes. 4. A sequence comparison for both animal and plant lipoxygenases shows strongly conserved amino acids, especially histidines and hydrophobic residues, which possibly may be involved in iron complexation and substrate binding.
Höhne
W E
WE
Institute of Biochemistry, Humboldt University of Berlin, FRG.
Kojima
N
N
Thiele
B
B
Rapoport
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002614:Chelating Agents / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D008084:Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors; D008322:Mammals; D008628:Mercury / Q000494:pharmacology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid; D013025:Glycine max / Q000201:enzymology*
D002614:Chelating Agents; D006146:Guanidines; D016859:Lipoxygenase Inhibitors; D010940:Plant Proteins; D007501:Iron; D008084:Lipoxygenase; D008628:Mercury; D019791:Guanidine