Characterization of two antioxidant enzymes: Paraoxonase-1, and peroxiredoxin-6

This study examines two antioxidant enzymes - paraoxonase-1 (PON1) and peroxiredoxin6 (prdx6).;PON1 is Ca2+ dependent glycoprotein that is associated with high-density lipoproteins (HDL). Although its physiological role is uncertain, it has been demonstrated to be a multifunctional enzyme. One of its functions implicates it to be important in the metabolism of phospholipid and cholesterol ester hydroperoxides, thereby preventing the oxidation of LDL and offering protection against atherosclerosis. There have been a number of reports that claim PON1 to function as a cysteine peroxidase by reducing oxidized lipoproteins found in oxidatively modified LDL particles. Results obtained thorough my studies, however, impugn the evidence that have suggested PON1 to function as a peroxidase. Using results from kinetic experiments, this study proposes that PON1 does not possess a peroxidative activity. It provides the first evidence that suggests that the peroxidase activity historically attributed to PON1 is due to an enzymatic impurity (probably a peroxidase) present in human serum and copurifying with PON1. This work adds a critical component to the growing body of PON1 research, all of which have slowly but incrementally chipped away at the reputation of PON1 functioning as a peroxidase. It not only implicates an enzymatic impurity to be responsible for the peroxidative activity but provides evidence that suggests the presence of an interaction between PON1 and the impurity.;Prdx6 is a bifunctional enzyme with both glutathione (GSH) peroxidase and phospholipase A2 (PLA2) activities. It is postulated that it functions in antioxidant defense by facilitating the repair of oxidatively damaged cell membranes by the reduction of peroxidized phospholipids. The peroxidase activity of prdx6 is thought to be catalyzed by C47 which initiates the nucleophilic attack. However, examination of the crystal structure of human prdx6 reveals C47 to be embedded inside the enzyme core, and thereby inaccessible to a lipid hydroperoxide substrate. It is assumed that the enzyme has to undergo a conformational change to function as a competent peroxidase. Yet, we have no evidence as to the nature of such a conformation. This work is an attempt to solve the structure of prdx6 bound to a PLA2 transition-state inhibitor, 1-hexadecyl-3-(trifluoroethyl)- sn-glycero-2-phosphomethanol lithium (MJ33) to gain insight into structural changes upon binding the inhibitor, and consequently, propose a mechanism for its peroxidative and PLA2 activities. Success has been obtained for the overexpression, purification, and characterization of both mutant and wild type forms of human and rat prdx6. These results indicate that prdx6 is stably overexpressed in bacterial cells. The purified enzyme is highly pure and homogeneous and aqueously soluble up to at least 18 mg/mL. Crystallization trials have yielded small needle-like crystals that diffract to a resolution of 3.5 A. Attempts to manipulate the quality of the crystals for increased resolution have proved unsuccessful. It is proposed that MJ33 might not be a suitable candidate for crystallization because it leads to a relatively rapid precipitation of prdx6 in crystallization trays. Further attempts at crystallization will employ the addition of different PLA2 inhibitors.