Adam OffenbacherJakobowski, Andrew Charles2025-06-122025-05May 2025May 2025http://hdl.handle.net/10342/14090Abstract: Lipoxygenases (LOXs) are a family of iron enzymes that catalyze the peroxidation of polyunsaturated fatty acids to generate oxylipins, which in both plants and animals play essential roles in signaling. In humans, LOX function presents a double-edged sword as they have known involvement in resolving and promoting chronic inflammation. Of the six LOX genes, human 15-lipoxygenase-2 (15-LOX-2) enzyme oxidizes arachidonic acid (AA) to produce 15-hydroperoxyeicosatetraenoic acid (15-HPETE) a precursor of lipoxins and correlated with plaque formation in arteries, increasing risk of cardiovascular disease. Despite related health impacts of 15-LOX-2, there are few selective inhibitors and no drugs prescribed in the United States that target and inhibit LOXs function. Additionally, the structural regulation of LOX function remains to be understood. Allosteric regulation provides a new outlook on the functional mechanism of proteins. Proteins bind a regulatory molecule or metal at a site other than the active site causing a conformational change that alters the protein’s activity. X-ray structures of 15-LOX-2 and other animal LOXs provide evidence of binding sites for divalent calcium ions (Ca2+) that are predicted to serve as allosteric effectors promoting full activity of animal LOXs in the presence of membrane causing the helix-α2 – the gatekeeper for substrate binding – to promote substrate acquisition to the active site. In this thesis, biochemical and biophysical methods such as isothermal titration calorimetry, differential scanning calorimetry, limited proteolysis, and surface plasmon resonance are used to understand the thermodynamic profiles that drive Ca2+ binding to promote protein-membrane interactions of 15-LOX-2 and thereby influence LOX catalysis.application/pdfEnglishChemistry, BiochemistryMaster's Thesis2025-05-22