MECHANISMS THAT REGULATE ENZYMES THAT METABOLIZE CIS UNSATURATED FATTY ACIDS

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Summary of Work: The goal is to develop an understanding of the biochemical mechanisms responsible for the metabolism of arachidonic acid and linoleic acid by these enzymes. Nitric oxide (NO) is reported to enhance prostaglandin formation by enhancement of PGHS activity. We found that NO is a substrate for the peroxidase of PGHS-1 and -2, but it does not enhance the cyclooxygenase activity in vitro. In macrophages, NO does not stimulate the expression of PGHS or alter LPS dependent PGHS-2 expression. Furthermore, NO did not enhance prostaglandin formation in intact cells. We propose htat peroxynitrite, a reaction product of NO and superoxide oxide is responsible. Other studies with PGHS-1 indicate that a tyrosyl radical is formed which could be the reactive intermediate that initiates arachidonic acid oxygenation. Recently, we have shown that NO reacts with the tyrosyl radical converting the radical to a new radical characterized by ESR analysis as an iminoxyl radical. The iminoxyl radical decays to nitrotyrosine resulting in the nitration of the tyrosine moiety on PHS. In addition, its formation of nitrotyrosine is often used as an indicator of oxidative stress. We have identified the specific tyrosine moiety in PHS nitrated by the reaction of NO with tyrosyl radical as Tyrosine-385. This indicates that tyrosine-385 is oxidized to tyrosyl radical during catalysis and suggests this residue is a likely active site or is the site of enzyme inactivation. Understanding the biochemical mechanisms which regulate the activities and expression of these enzymes will provide new insights into potentially controlling or preventing these disease conditions.