The overall goal is to provide a molecular understanding of enzymic mechanism of Prostaglandin H synthase (PGHS) and endothelial type nitric oxide synthase (eNOS). 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 propagation of the cyclooxygenase reaction. The testing hypothesis for eNOS is a 3/2 coupling model( 3 cycles of NADPH oxidation with 2 cycles of P450 catalysis) between the reductase and the P450 oxidase mediated by CaM/Ca2+ and tetrahydrobiopterin (H4B). To test these mechanistic models, we propose to: 1. Carry out detail studies of the PGHS cyclooxygenase catalytic cycle, and kinetic characterization of the self-inactivation using stopped-flow, rapid freezing and rapid quenching methods. 2. Characterize the structure-activity relationship between PGHS and different nonsteroidal anti-inflammatory agents (NSAIDS) based on their effect in perturbing the conformation of a key tyrosyl radical, which mediates the peroxidase and cyclooxygenase cycles. EPR and molecular modeling will be employed in this study. 3. Characterize the eNOS heme using whole eNOS molecule or individual domains of reductase and oxidase obtained by limited trypsin digestion. Transient kinetic measurements will be performed and the effect of CaM/Ca2+ and H4B will be evaluated in the context of kinetics. The first specific approach helps to gain knowledge about the individual steps of the cycloooxygenase catalysis and the rates of self-inactivation. The second study will provide useful information about the pharmacological action of NSAIDS on PGHS and advance the knowledge of drug design. The third approach will help in defining the detail chemical and physical properties of eNOS and obtain valuable information about the structure of the heme center. The last approach will lay solid ground in the elucidation of the reaction mechanism of this complicated self- sufficient NADPH cytochrome P450 systems. Understanding the basic reaction mechanism of these two key enzymes in the cardiovascular systems should cast great insight into the control and regulation of the physiological and pathophysiological events associated with prostaglandins and nitric oxide.
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