Tyrosine 385 of prostaglandin endoperoxide synthase is required for cyclooxygenase catalysis.
There are spectral and biochemical data suggesting that a tyrosine group(s) is involved in the cyclooxygenase reaction catalyzed by prostaglandin endoperoxide (PGH) synthase. Treatment with tetranitromethane, a reagent which nitrates tyrosine residues, abolishes cyclooxygenase activity, but this inactivation can be largely prevented by competitive cyclooxygenase inhibitors such as ibuprofen and indomethacin. To identify sites of nitration, native PGH synthase and indomethacin-pretreated PGH synthase were incubated with tetranitromethane, and the sequences of peptides containing nitrotyrosine were determined. Three unique tyrosines (Tyr-355, Tyr-385, and Tyr-417) were nitrated in the native enzyme but not in the indomethacin-treated PGH synthase. Using site-directed mutagenesis of sheep PGH synthase, each of these tyrosines, as well as two other tyrosine residues selected as controls (Tyr-254 and Tyr-262), were replaced with phenylalanine; cos-1 cells were transfected with constructs containing cDNAs coding for the native PGH synthase and each of the five phenylalanine mutants, and microsomes from these cells were assayed for cyclooxygenase and hydroperoxidase activities. The Phe-385 mutant of PGH synthase lacked cyclooxygenase activity but retained peroxidase activity; all other mutants expressed both enzyme activities. Our results establish that Tyr-385 is essential for the cyclooxygenase activity of PGH synthase and that nitration of this residue can be prevented by indomethacin. We conclude that Tyr-385 is at or near the cyclooxygenase active site of PGH synthase and could be the tyrosine residue proposed to be involved in the first step of the cyclooxygenase reaction, abstraction of the 13-proS hydrogen from arachidonate.
Shimokawa
T
T
Department of Biochemistry, Michigan State University, East Lansing 48824.
Kulmacz
R J
RJ
DeWitt
D L
DL
Smith
W L
WL
D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D007213:Indomethacin / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D012756:Sheep; D013774:Tetranitromethane / Q000494:pharmacology; D014162:Transfection; D014443:Tyrosine
D001095:Arachidonic Acids; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D014443:Tyrosine; D011451:Prostaglandin-Endoperoxide Synthases; D013774:Tetranitromethane; D007213:Indomethacin