Date of Thesis
Soybean Lipoxygenase-1 (SBLO-1) is proposed to bind to its fatty acid substrate, linoleic acid, in a "tail-first" manner, where the methyl terminus of linoleic acid binds near phe-557 in the active site. In order to test the "tail-first" theory of binding, a substrate with two additional carbons on its tail end, 9,12 eicosadienoic acid (9,12 EDA) was synthesized. The starting compounds for the synthesis of 9,12 EDA were decyne and 8-bromooctanoic acid. High pressure liquid chromatography (HPLC) was used to purify the 9,12 EDA substrate, which was used in enzymatic experiments with wild-type (WT) and F557A SBLO-1. With WT SBLO-1, the addition of two carbons to linoleic acid causes the regiochemistry of the reaction to shift from 96% carbon-13 oxygenation to 48% carbon-13 oxygenation. With the F557A mutant, the addition of two carbons to linoleic acid causes the regiochemistry of the reaction to shift, to a lesser extent, from 96% carbon-13 oxygenation to 67% carbon-13 oxygenation. With both WT and F557A SBLO-1, the 9,12 EDA substrate demonstrated stereospecificity. Kinetic experiments with 9,12 EDA showed a significant induction period at high substrate concentrations, which was corrected for with the addition of 2 μM 13-hydroperoxydecadienoic acid (13-HPOD). Michaelis-Menten kinetics with 9,12 EDA gave kcat/Km (s-1µM-1) values of 8.0 ± 2.0 and 13.0 ± 5.0, respectively for WT and F557A SBLO-1. With WT enzyme, adding two carbons to linoleic acid results in a two-fold reduction in the rate of oxygenation at low substrate concentration. With F557A enzyme, adding two carbons to linoleic acid does not affect the rate of oxygenation at low substrate concentration. The addition of two extra carbons to linoleic acid impedes proper substrate binding to the WT enzyme, and reducing the size of residue 557 makes it easier for SBLO-1 to catalyze its normal reaction on the longer substrate. The results are consistent with the "tail-first" binding hypothesis.
Honors Thesis (Bucknell Access Only)
Bachelor of Science in Biomedical Engineering
Charles Himes Clapp
Hinnenkamp, David Joseph, "Synthetic Fatty Acids as Probes for Substrate Binding by Soybean Lipoxygenase-1" (2017). Honors Theses. 386.