Date of Thesis

4-27-2015

Thesis Type

Honors Thesis (Bucknell Access Only)

Degree Type

Bachelor of Science

First Advisor

Charles H. Clapp

Abstract

Soybean lipoxygenase-1 (SBLO-1) is a non-heme iron containing enzyme that catalyzes the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Even though the catalytic mechanism is partially understood, it is still unclear how the substrate binds to the enzyme. It is proposed that the major product is produced by tail-first binding, while the minor product is produced by head-first binding. Phenylalanine-557, a key residue proposed to be involved in substrate binding, was replaced by valine in order to test the binding modes. The phenylalanine-557-valine (F557V) mutant was studied with both natural substrates and synthetic substrates in order to probe key features of the active site. Product analyses of linoleic acid with F557V confirms that the elevated production of 9-hydroperoxy-10(E), 12(Z)-octadecadienoate (9-HPOD) occurs at low substrate concentrations, consistent with previous work done at high substrate concentrations. The product analyses of linoleic acid, arachidonic acid and linoleoyl-D-tryptophan supports the proposed binding modes that lead to major product and minor product. Kinetic studies of linoleic acid show that kcat for F557V is significantly lower than that for the wild-type SBLO-1. Severe substrate inhibition was observed with 9(Z),12(Z)-nonadecadienoic acid (9,12-NDA) but not with (10Z,13Z)-10,13-nonadecadienoic acid (10,13-NDA). However, the product analyses of 9,12-NDA do not support the hypothesis that phynylalanine-557 is at the binding site for the methyl terminus of the substrate. Thus the binding mechanism could be more complicated than the two binding modes that are proposed.

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