Date of Thesis

2017

Description

Lipoxygenases are iron-containing enzymes that catalyze the peroxidation of polyunsaturated fatty acids containing cis double bonds. The catalytic mechanism of soybean lipoxygenases (SBLO-1) has been extensively studied, but the mode of substrate entry into the binding site remains poorly understood. It is widely proposed that substrates enter the active site of SBLO-1 through tail-first or head-first binding, and that phenylalanine at the 557th position (F557) might play a role in determining which method is utilized. To understand its potential influence on substrate binding, this residue was replaced with alanine (F557A), serine (F557S), and valine (F557V). Product analysis of linoleic acid oxygenation by these mutants indicates an increase in production of 9 (S)-hydroxy-10(E),12 (Z)-octadecadieonic acid for F557V while the product ratio for F557A and F557S is similar to that of the wild-type. Analysis of the F557A products also indicates tail-first binding to be the main binding method for 13 (S)-hydroxy-9(Z),11 (E)-octadecadieonic acid formation. The F557V reaction with 2-linoleoyl-1-palmitoyl-sn-glycero-3-phosphocholine indicates that head-first binding for this substrate is not likely to occur due to the size and polarity of the head group. Product analysis of the reaction between 9(Z), 12(Z)-nonadecadienoic acid (9, 12-NDA) and F557A indicates that the mutation did not improve the ability of SBLO-1 to oxygenate the substrate at carbon-13. Finally, kinetic studies of 9, 12-NDA with the wild-type and F557A mutant reveal that 9, 12-NDA is a poorer substrate than linoleic acid. These results indicate that the mode of substrate binding cannot be predicted simply by the size of the residue at position 557.

Access Type

Honors Thesis (Bucknell Access Only)

Degree Type

Bachelor of Science

Major

Cell Biology/Biochemistry

First Advisor

Charles Himes Clapp

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