The cyclooxygenase activity of prostaglandin H synthase (PGHS) catalyzes the first committed step in the biosynthesis of the prostaglandins, a group of potent, bioactive lipids believed important in many pathophysiological processes, including inflammation, vascular, gastric and renal function, reproduction, and tumorigenesis. Two PGHS isoforms are known: PGHS-1 is regarded as constitutive and is ascribed housekeeping function; PGHS 2 is strongly inducible by cytokines in many cells involved in inflammatory and proliferative processes. Besides the controls of PGHS- 1 and -2 gene expression, cellular prostaglandin synthesis is also tightly regulated at the cyclooxygenase catalytic level, with different catalytic controls for the two PGHS isoforms. PGHS-2 cyclooxygenase has a much lower hydroperoxide activator requirement than the PGHS-1 cyclooxygenase. This difference in feedback activation by the product provides a simple biochemical basis for differential cellular control of cyclooxygenase catalysis. Cellular cyclooxygenase catalysis also can be limited by fatty acid substrate availability. PGHS-1 cyclooxygenase activity exhibits cooperative behavior at low arachidonic acid levels, whereas the PGHS-2 enzyme follows simple saturable kinetics. 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 and targeted mutant proteins to achieve the following specific aims: 1) Identify the mechanistic basis(es) for the different hydroperoxide activator requirements in PGHS-1 and -2, and investigate the mechanistic changes in aspirin-treated PGHS-2; 2) Identify the structural element(s) controlling the hydroperoxide activator requirements in PGHS-1 and -2, and investigate the mechanistic changes in aspirin-treated PGHS-2; 2) Identify the structural element(s) controlling the hydroperoxide activator requirements in the two isoforms; and 3) Evaluate the interdependence between fatty acid and peroxide in cyclooxygenase catalytic control for the two PGHS isoforms.
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