PMID: 2122967Nov 25, 1990Paper

Tyrosine 385 of prostaglandin endoperoxide synthase is required for cyclooxygenase catalysis.

The Journal of Biological Chemistry
T ShimokawaW L Smith

Abstract

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 ...Continue Reading

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