Date of Thesis
Masters Thesis (Bucknell Access Only)
Master of Science
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.
Hershelman, Dillon J., "Probing of Head-first Substrate Binding to the Phe557Val Mutant of Soybean Lipoxygenase-1 with 11(S)-Deuterated Linoleic Acid" (2018). Master’s Theses. 213.