An examination of the source of the tyrosyl radical in ovine prostaglandin endoperoxide synthase-1.
A tyrosyl radical, which may initiate the cyclooxygenase reaction, has been detected in prostaglandin H synthase by electron paramagnetic resonance spectroscopy. In the crystal structure of ovine prostaglandin H synthase-1, Tyr348 and Tyr385 are in close proximity to the heme. We mutated these residues to phenylalanine to test for their involvement in tyrosyl radical formation. Native enzyme formed a tyrosyl radical centered at g = 2.0036 with a width of 28 gauss. The Y348F mutant formed a singlet signal similar to that of native enzyme with a width of 28 gauss (g = 2.0039). In contrast, the radical signals seen with the Y385F and Y348F/Y385F mutants were 23 gauss (g = 2.004) and 22 gauss (g = 2.0037). In short, tyrosyl radicals are formed even in the absence of both Tyr348 and Tyr385. In Y345F containing mutants, a cluster of aromatic amino acids which surrounds the heme group may provide an alternate pathway for electron abstraction from a more distant tyrosine, yielding a narrow tyrosyl radical signal.
Hsi
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Department of Biochemistry, Michigan State University, East Lansing 48824.
Hoganson
C W
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Babcock
G T
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Garavito
R M
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Smith
W L
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000032:analysis / Q000378:metabolism; D006418:Heme / Q000032:analysis; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D017354:Point Mutation; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D012756:Sheep; D014443:Tyrosine
D000906:Antibodies; D005609:Free Radicals; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011994:Recombinant Proteins; D014443:Tyrosine; D006418:Heme; D011451:Prostaglandin-Endoperoxide Synthases