IDENTIFICATION OF A SPECIFIC METHIONINE IN MAMMALIAN 15-LIPOXYGENASE WHICH IS OXYGENATED BY THE ENZYME PRODUCT 13-HPODE - DISSOCIATION OF SULFOXIDE FORMATION FROM SELF-INACTIVATION

Mammalian 15-lipoxygenases undergo a characteristic self-inactivation. The oxygenation of a single methionine to methionine sulfoxide, by 13 (S)-hydroperoxyoctadecadienoic acid (13-HPODE), was previously suggest ed as the cause of the inactivation of rabbit reticulocyte lipoxygenas e. The site of oxygenation is potentially near the enzyme's active sit e; however, the specific location of the modified amino acid residue h as not been identified. To determine which of the methionine residues is oxygenated, we inactivated both human and rabbit 15-lipoxygenases w ith 13-HPODE and sequentially denatured, reduced, carboxymethylated, a nd digested the enzymes with trypsin. The digested mixtures were analy zed by reverse-phase HPLC chromatography. Mass spectrometric analysis of each of the methionine-containing fractions enabled us to locate th e peptide segments containing the oxidized methionine in both enzymes separately. Tandem electrospray mass spectrometry identified the oxidi zed methionine residues to be amino acid 590 in the human enzyme and 5 91 in the rabbit enzyme. To investigate the significance of this oxyge nation, Met(590) in human 15-lipoxygenase was substituted with leucine by site-directed mutagenesis. The mutant protein was inactivated by 1 3-HPODE, yet no oxygenated peptide or other modified peptide could be identified by HPLC-MS analysis. We also found that human 15-lipoxygena se was inactivated during arachidonate oxidation and by the reaction p roduct 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), and no modif ied peptide was detected. Thus, methionine oxygenation is not essentia l for the inactivation of human 15-lipoxygenase. We suggest, however, that Met(590) is an amino acid in the substrate binding pocket of huma n 15-lipoxygenase and interacts with the enzyme product 13-HPODE.