Kinetic investigation of oxidized polyunsaturated fatty acids as inhibitors of human serum paraoxonase
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Atherosclerosis and coronary vascular disease are the major causes of morbidity and mortality in the western world. The oxidative modification of low-density lipoprotein (LDL) is central to the pathogenesis of atherosclerosis. High-density lipoprotein (HDL) retards the accumulation of lipid peroxides when LDL is incubated under oxidizing conditions. This protective effect is primarily due to the activity of the enzyme paraoxonase 1 (PON1) which is simultaneously inhibited by the products of lipid peroxidation. PON1 has a functional polymorphism known as the Q192R polymorphism and is exclusively associated with HDL in the circulation where PON1 activity has been shown to be inversely related to the risk of coronary heart disease, hypercholesterolemia, and diabetes. The objectives of this thesis are to characterize the inhibition kinetics of isolated and purified human serum PON1 alloenzymes by oxidatively modified lipids. This was performed, first by synthesizing oxidized lipids by oxidizing commercially purchased polyunsaturated fatty acids; Second PON1 was isolated and purified from Serum; Third, inhibition kinetics was performed using the isolated and purified PON1 and the oxidized lipids: Lastly we quantified the kinetic inhibition as either: competitive, uncompetitive, noncompetitive, and/or mixed competitive. Our results indicate that PON1 is inhibited by oxidized polyunsaturated fatty acids in a mixed competitive manner. These results support the supposition, derived from the three dimensional structure of PON1 reported during the time period that we were performing our research, that PON1 is an interfacial enzyme within the HDL particle.