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 suggested as the cause of the inactivation of rabbit reticulocyte lipoxygenase. The site of oxygenation is potentially near the enzyme's active site; however, the specific location of the modified amino acid residue has not been identified. To determine which of the methionine residues is oxygenated, we inactivated both human and rabbit 15-lipoxygenases with 13-HPODE and sequentially denatured, reduced, carboxymethylated, and digested the enzymes with trypsin. The digested mixtures were analyzed by reverse-phase HPLC chromatography. Mass spectrometric analysis of each of the methionine-containing fractions enabled us to locate the peptide segments containing the oxidized methionine in both enzymes separately. Tandem electrospray mass spectrometry identified the oxidized methionine residues to be amino acid 590 in the human enzyme and 591 in the rabbit enzyme. To investigate the significance of this oxygenation, Met590 in human 15-lipoxygenase was substituted with leucine by site-directed mutagenesis. The mutant protein was inactivated by 13-HPODE, yet no oxygenated peptide or other modified peptide could be identified by HPLC-MS analysis. We also found that human 15-lipoxygenase was inactivated during arachidonate oxidation and by the reaction product 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), and no modified peptide was detected. Thus, methionine oxygenation is not essential for the inactivation of human 15-lipoxygenase. We suggest, however, that Met590 is an amino acid in the substrate binding pocket of human 15-lipoxygenase and interacts with the enzyme product 13-HPODE.
Gan
Q F
QF
Syntex Discovery Research, Palo Alto, California 94304, USA.
Witkop
G L
GL
Sloane
D L
DL
Straub
K M
KM
Sigal
E
E
D015117:5,8,11,14-Eicosatetraynoic Acid / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006801:Humans; D007501:Iron / Q000032:analysis; D015289:Leukotrienes / Q000737:chemistry; D008041:Linoleic Acids / Q000737:chemistry*; D008054:Lipid Peroxides / Q000737:chemistry; D016859:Lipoxygenase Inhibitors; D013058:Mass Spectrometry; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010100:Oxygen / Q000737:chemistry*; D010449:Peptide Mapping; D011817:Rabbits; D014357:Trypsin
D015289:Leukotrienes; D008041:Linoleic Acids; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; D009838:Oligodeoxyribonucleotides; D015117:5,8,11,14-Eicosatetraynoic Acid; C038649:13-hydroperoxy-9,11-octadecadienoic acid; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D008715:Methionine; D007501:Iron; D001093:Arachidonate 15-Lipoxygenase; D014357:Trypsin; D010100:Oxygen; C013111:methionine sulfoxide