Characterization of the tyrosyl radicals in ovine prostaglandin H synthase-1 by isotope replacement and site-directed mutagenesis.
Several free radical species, attributed to tyrosyl radicals, have been previously observed by electron paramagnetic resonance (EPR) spectroscopy during reaction of pure ovine prostaglandin H synthase-1 (PGHS-1) with hydroperoxide or fatty acid substrates. The identity and location of amino acid residue(s) involved in formation of these radicals has been investigated using wild type and mutant PGHS-1 expressed in transfected COS-1 cells. Upon reaction with hydroperoxide, detergent extracts from microsomes of cells expressing wild type ovine PGHS-1 rapidly produced EPR signals made up of a broad component, resembling either the wide doublet or the wide singlet seen in reactions of the purified ovine enzyme, mixed with a variable proportion of a narrow component. Isotope replacement with perdeuterated tyrosine and phenylalanine in wild type PGHS-1 caused no change in enzymatic activity or capability to form a peroxide-induced radical, but the radical was an isotropic singlet without any wide features. This loss of splitting in the deuterated enzyme establishes that the wide radical signal in PGHS-1 is indeed a tyrosyl radical, validating the earlier assignment based on spectral similarities with a tyrosyl radical in ribonucleotide reductase. A Y385F mutation abolished cyclooxygenase but not peroxidase activity, and the peroxide-induced EPR was narrowed to a singlet essentially identical with that obtained with indomethacin-treated ovine enzyme. Isotopic replacement with perdeuterated tyrosine and phenylalanine in the Y385F mutant led to further narrowing of the peroxide-induced EPR to an isotropic singlet, establishing that this radical also involved a tyrosine residue. A Y355F mutant exhibited some decrease in cyclooxygenase activity, but the peroxidase activity and the peroxide-induced radical characteristics were not significantly different from those of the wild type enzyme, indicating that Tyr355 is not involved in tyrosyl radical formation in PGHS-1.(ABSTRACT TRUNCATED AT 400 WORDS)
Tsai
A
A
Department of Internal Medicine, University of Texas Health Science Center, Houston 77030.
Hsi
L C
LC
Kulmacz
R J
RJ
Palmer
G
G
Smith
W L
WL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000032:analysis; D015870:Gene Expression; D006861:Hydrogen Peroxide / Q000378:metabolism; D007213:Indomethacin / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012756:Sheep; D013379:Substrate Specificity; D014162:Transfection; D014443:Tyrosine / Q000032:analysis*
D005609:Free Radicals; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014443:Tyrosine; D006861:Hydrogen Peroxide; D011451:Prostaglandin-Endoperoxide Synthases; D007213:Indomethacin