FUNCTIONAL STUDIES OF HUMAN AND SOYBEAN LIPOXYGENASE

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DESCRIPTION: Lipoxygenases (LOs) are enzymes that catalyze the incorporation of dioxygen into 1,4-cis,cis-pentadiene containing fatty acids, and are widely distributed throughout the plant and animal kingdoms. The activity of the enzyme has been implicated in plant germination, human asthma and arthritis. The active site of the enzyme contains an essential iron atom. Two distinct mechanisms for LO catalysis have been proposed. The first involves a radical based mechanism in which the active site ferric ion oxidizes the 1,4-diene of linoleic acid (LA) to a fatty acid radical. The second mechanism uses the ferric iron to facilitate de-protonation of the substrate by coordinating the resulting carbonation to form an organometallic intermediate. Thus, the key difference in the two mechanisms is the role of the iron center and how it activates the proton abstraction. This proposal involves spectroscopic and kinetic studies aimed at investigating the structure and function of the active site iron in both soybean and human lipoxygenases. In addition, a number of active site mutants designed to affect the electronic structure of the iron and its reactivity will be studied. Spectroscopic techniques (e.g., EPR, MCD, EXAFS) will be used to determine the changes in the iron coordination and/or electronic environment. These studies will be correlated to kinetic investigations that address the effect of the structural changes on reactivity as well as the effects of pH, viscosity and isotopic substitutions. The information obtained will be used to determine a unified theory of the structure and function of the Fe center in catalyzing the oxidation reactions.