Structure/Function and Reaction Mechanism of PGHS

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DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate structure/function relationships and reaction mechanism of prostaglandin H synthase (PGHS), which plays key roles in vascular physiology and pathophysiology. PGHS has two distinct enzyme activities: a cyclooxygenase that converts arachidonic acid (AA) to PGG2, and a peroxidase that reduces PGG2 to PGH2. A free radical branched-chain mechanism involving a key tyrosyl radical and substrate-derived radicals will be tested to elucidate the self-activation properties of PGHS catalysis. Three specific aims are included in this application. We will characterize the structure and kinetics of the AA-derived pentadienyl and allyl radical using specifically isotope labeled AA by rapid-freeze EPR, ENDOR in the first aim. Primary and secondary KIE will be employed for the H-abstraction and allyl radical formation. The second aim is to test the hypothesis that either tyrosyl radical migration or/and the tyrosyl radical phenyl ring rotation plays a decisive role in coupling the peroxidase to cyclooxygenase catalysis. We will differentiate the tyrosyl radical phenyl ring rotation mechanism from radical migration by minimizing the latter mechanism using single and multiple tyrosine mutants to locate the alternative tyrosine that participate in radical migration. Correlative kinetic measurements will be done between tyrosyl radical conformation dynamics with peroxidase and cyclooxygenase catalysis. EPR, ENDOR, and HFEPR will examine freeze-trapped tyrosyl radical at different conformation. To achieve the last aim, heme orientation relative to the microsome membrane surface will be determined by linear dichroism and EPR on the oriented membrane to address the issue on substrate presentation, product release and the communication between PGHS and downstream enzymes in eicosanoid biosynthesis. The 1st Aim is to identify additional intermediate(s), to elucidate the structure variation of AA-derived radical and the mechanism of the cyclooxygenase catalysis. The 2nd Aim will address the role of tyrosyl radical dynamics on PGHS catalysis. The last aim will provide direct data on the membrane topology of PGHS and the mechanism of its substrate presentation and product release.