Date of Thesis
Lipoxygenases are a class of non-heme iron-containing enzymes involved in the oxygenation of polyunsaturated fatty acids. In animals, lipoxygenases are involved in the synthesis of precursor molecules that are involved in cell membrane maturation, cancer metastasis, and the biosynthesis of leukotrienes that are involved in the inflammatory response. These enzymes are important in a variety of human diseases, including asthma, arthritis, and cancer. The mechanism of substrate binding to mammalian lipoxygenase (15-LOX) is unknown, but the enzyme is unstable and difficult to study. Soybean lipoxygenase-1 (SBLO-1) shares significant homology and structure with 15-LOX, making it a suitable replacement for studies. The goal of this work was to test the hypothesis that binding of the substrate, linoleic acid, to SBLO-1 requires transient separation of helices 2 and 11. In this study, a disulfide bond-forming double mutant (T259C, S545C) has been made to prevent the separation of these two helices. SDS-PAGE and mass spectrometry were completed to determine under which conditions the disulfide bond forms. UV/Vis spectrophotometry activity assays were performed to calculate the specific activity of this mutant enzyme under oxidizing and reducing conditions to determine what effect the formation of a disulfide bond had on catalytic activity. These studies indicate that the formation of a disulfide bond does not substantially reduce the catalytic activity of the T259C, S545C SBLO-1 mutant. This result demonstrates that linoleic acid binding to SBLO-1 does not require complete separation of helices 2 and 11. Instead, it is possible that these helices are quite flexible and only part of the helices must separate for the substrate to gain access to the active site.
protein biochemistry, mass spectrometry, disulfide crosslinking
Honors Thesis (Bucknell Access Only)
Bachelor of Science in Chemical Engineering
Charles Himes Clapp
Erin L. Jablonski
Milligan, Madeline Patricia, "Disulfide Crosslinking Studies to Probe Substrate Binding to Soybean Lipoxygenase-1" (2017). Honors Theses. 414.