The overall goal is to provide a molecular understanding the enzyme reaction mechanism of Prostaglandin H synthase (PGHS). The working model for PGHS is a free radical branched chain mechanism in which a tyrosine radical, generated after interaction of the synthase heme with hydroperoxide in the peroxidase catalytic cycle, serves a central catalytic role in the initiation and propagation of the cyclooxygenase reaction. This same tyrosyl radical also serves as the key intermediate to launch the self-inactivation of PGHS. To test these mechanistic models, we propose to: 1) Carry out detailed studies of the PGHS cyclooxygenase catalytic cycle using series of specific-labeled arachidonic acid. Rapid-freeze/EPR and rapid-quenching/HPLC methods will be employed to define the rates and to determine the structure of the main intermediates and enzyme products. Characterize the mechanistic differences between the Fe-PGHS and PGHS reconstituted with Mn(II) or Mn(III) protoporphyrins. 2) Elucidate the mechanism of self-inactivation occurring in both the peroxidase and cyclooxygenase catalysis. Rapid-scan stopped-flow will be used to determine the rates and to identify the key intermediates. 3) Evaluate the structure-function relationships by site-directed mutagenesis. The key residues involved in the peroxidase catalysis, self-inactivation and the stability of the radical intermediates will be investigated. The first study helps to gain knowledge about the individual steps of cyclooxygenase catalysis and the rates of key reactions. The second study will provide useful information about the ultimate regulation of PGHS catalytic activity. The last study will pin point the key elements in the protein that play specific functional roles in catalysis and inactivation. Understanding the basic reaction mechanism of PGHS in the cardiovascular systems should provide great insight into the control and regulation of the physiological and pathophysiological events associated with prostaglandins.
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