Our constant exposure to oxygen radicals and related oxidants can initiate chronic and acute disease states associated with aging (e.g., atherosclerosis, arthritis, and cancer). Prostaglandin H synthase (PGHS)-1 and -2 are integral membrane heme-enzymes which convert arachidonic acid into the precursors of the prostaglandin cascade. The PGHS isoforms generate free radicals that damage cell and cell components and activate environmental contaminants. The PGHS peroxidase constitutes a distinct and complementary pathway to the structure is relevant to pathology of oxidative damage for two reasons. First, the tertiary structure of PGHS is not only homologous to that of other mammalian peroxidases but also to the large family of mammalian peroxidases. Second, an understanding of the enzyme's structure is a prerequisite for understanding the mechanism that generates oxidative products. The nature of enzyme-ligand interactions and structure/function relationships in the native PGHS peroxidases, as well as in site-directed mutants will be explored using stopped flow UV-Vis absorption spectroscopy, resonance Raman spectroscopy and X-ray crystallography.
The Specific Aims are as follows. First, we will investigate the nature of enzyme-ligand interactions and associated structure-function relationships in the native peroxidase of PGHS isoforms to characterize the heme iron environment through the use of heme iron ligands and to identify and then characterize the binding site(s) for reducing substrates using weak (i.e., more stable) reductants such as 2-aminofluorene and benzyl hydroxamic acid. Second, we will study the structure/function relationships in the ovine PGHS-1 peroxidase via mutational analyses in the peroxidase site. Third, we will study the structure-function relationships in the human PGHS-2 peroxidase by examining modified PGHS-2 with equivalent mutations as in ovine PGHS-1. Finally, we will attempt to follow the structural changes involved in free radical generation to elucidate the mechanism of free radical transfer and peroxidase-dependent inactivation. The goal is to identify and stabilize long-lived oxidative or free radical states created by the peroxidase reaction, in solution and in the crystal. A subsidiary goal is also to determine the nature of the free radical damage that leads to inactivation of the PGHS peroxidase and to develop a model for free radical damage of protein in other biological systems.
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