Date of Thesis

Summer 2018

Thesis Type

Masters Thesis (Bucknell Access Only)

Degree Type

Master of Science



First Advisor

Dr. Charles Clapp


Soybean Lipoxygenase, Deuterium, Kinetic Isotope Effect, Stereoselective, HPLC, Electrospray Mass Spectrometry


Soybean lipoxygenase-1 (SBLO-1) is an enzyme that abstracts the pro-S hydrogen at C-11 of linoleic acid and oxidizes it to predominantly 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate (13-HPOD) and a small amount of 9(S)-hydroperoxy-13(Z),11(E)-octadecadienoate (9-HPOD). The manner in which the substrate binds is uncertain, one hypothesis is that substrates bind primarily tail-first (with their methyl termini in the binding pocket) affording 13-HPOD products. Formation of 9-HPOD was described by Feussner et al. to arise from linoleic acid binding in a head-first manner (with its carboxylate terminus in the binding pocket). The Feussner binding model explains that SBLO-1 produces mostly 13-HPOD due to presence of phenylalanine at 557 in the binding pocket which natively coordinates the methyl terminus of linoleic acid leading to tail-first binding.

Previous research has demonstrated that while the wild-type of SBLO-1 (WT) produces 10% 9-HPOD at pH 7.5, the F557V mutant of SBLO-1 produces 37% 9-HPOD. The present research investigated this tail-first binding hypothesis using linoleic acid singly deuterated in the pro-S position on C-11 (11(S)-DLA). This substrate was obtained by chemically synthesizing racemic 11-deuteriolinoleic acid and treating it with SBLO-1, which selectively consumes the (R)-deuterated material due to the enzyme’s pro-S selectivity and the large primary kinetic isotope effect (KIE) of ~60 tied to deuterium abstraction.

When the 11(S)-DLA was used as a substrate for WT and the F557V mutant enzymatic reactions, the peroxy-products (13-HPOD and 9-HPOD) were reduced to alcohols (13-HOD and 9-HOD). The ratios of the 13 and 9-HOD products were analyzed by HPLC, the products themselves were purified via HPLC, and were analyzed for deuterium by electrospray mass spectrometry. The amount of 9-HPOD increased to 78% and 93% respectively. The shift in major enzymatic product was likely brought about by the large KIE linked to abstracting the deuterium in 11(S)-DLA. Nearly all of the 9-HOD (>98%) from either of the enzymatic experiments with 11(S)-DLA was deuterated. The results demonstrate that the formation of 9-HOD by SBLO-1 enzymes involves binding linoleic acid in a manner different from normal binding and removal of the pro-R hydrogen, which supports the Feussner binding model that predicts 9-HOD results from reverse binding.