Inactivation of human manganese-superoxide dismutase by peroxynitrite is caused by exclusive nitration of tyrosine 34 to 3-nitrotyrosine.
Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O-2 gateway in Mn-SOD.
Yamakura
F
F
Department of Chemistry, Juntendo University School of Medicine, Inba, Chiba 270-16, Japan. yamakura@sakura.juntendo.ac.jp
Taka
H
H
Fujimura
T
T
Murayama
K
K
D000595:Amino Acid Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D009566:Nitrates / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009566:Nitrates; D010446:Peptide Fragments; D011994:Recombinant Proteins; C039642:peroxynitric acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D013482:Superoxide Dismutase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase