The cyclooxygenase activity of prostaglandin H synthase (PGHS) catalyzes the first committed step in biosynthesis of the prostaglandins, a group of potent bioactive lipids important to many pathophysiological processes, including inflammation, vascular, gastric and renal function, reproduction, and tumorigenesis. PGHS is a member of the myeloperoxidase family and two PGHS isoforms are known: PGHS-1 is generally regarded as constitutive, with housekeeping functions; PGHS-2 is strongly inducible by cytokines in many cells involved in inflammatory and proliferative processes. Besides controls on PGHS-1 and -2 gene expression, cellular prostaglandin synthesis is also tightly regulated at the cyclooxygenase catalytic level, with quite distinct catalytic controls for the two PGHS isoforms. PGHS-2 cyclooxygenase has a much lower hydroperoxide activator requirement than PGHS-1 cyclooxygenase. This difference in feedback activation by product provides a simple biochemical basis for the differential cellular control of cyclooxygenase catalysis via suppressive actions of cellular peroxidases on peroxide levels. Peroxide activator is used to form a tyrosyl radical in the cyclooxygenase active site; this radical forms faster and is more stable in PGHS-2 than in PGHS-1. The general goal of this project is to understand the regulation of catalysis by the PGHS isoforms at a molecular level. Kinetic, spectroscopic, and structural studies will be undertaken with the two PGHS isoforms, another fatty acid oxygenase from the myeloperoxidase family, and targeted mutant proteins to achieve the following specific aims: 1) Characterize the PGHS-2 structural features governing tyrosyl radical formation, stability, and destructive side reactions and identify the structural basis for the higher cyclooxygenase activation efficiency in PGHS-2; 2) Characterize the effects of a membrane environment on PGG2 channeling in PGHS-I and -2 and on suppressive actions of phospholipid hydroperoxide glutathione peroxidase (HGPx) and cytosolic glutathione peroxidase (cGPx); and 3) Evaluate the generality of mammalian PGHS reaction mechanisms and catalytic regulation schemes using trout PGHS isoforms and plant pathogen-induced oxygenase (PIOX) as models.
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