CATALYTIC EFFICIENCY IN SOYBEAN LIPOXYGENASE-1

University Of California Berkeley, Berkeley CA

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Abstract

Lipoxygenases catalyze the insertion of molecular oxygen into poly- unsaturated fatty acids, making hydroperoxyacids. This is the first committed step in various metabolic pathways which lead to potent biological mediators, such as leukotrienes, which mediate the local inflammation response in bronchial constriction and are implicated in asthma. Understanding how these enzymes catalyze rapid chemical transformations is essential to the biomedical field. This research will explore those factors leading to a high rate of catalysis in Soybean Lipoxygenase-1 (LOX). Extensive quantum tunneling of the hydrogen nucleus occurs in the first step, a hydrogen abstraction which generates the reactive substrate radical (S). Site-directed mutagenesis of LOX will be performed to create mutants exhibiting reduced rate constants, in order to probe the relationship between catalytic efficiency and tunneling. Also, kinetic experiments using specifically labelled substrates will be performed to learn the identity of S. The O2 insertion occurs rapidly, but the mechanism of this insertion is not resolved. A series of rapid-mixing and steady-state kinetic experiments will be performed to identify possible intermediates between the formation of S and the release of product hydroperoxyacid, as well as to determine the limiting step in O2 insertion.