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Defining Protein Motions the Comprise the Reaction Barrier in Human Epithelial 15-Lipoxygenase-2

dc.access.optionOpen Access
dc.contributor.advisorOffenbacher, Adam R
dc.contributor.authorOhler, Amanda
dc.contributor.departmentChemistry
dc.date.accessioned2022-06-14T02:49:41Z
dc.date.available2024-05-01T08:02:29Z
dc.date.created2022-05
dc.date.issued2022-04-27
dc.date.submittedMay 2022
dc.date.updated2022-06-07T16:43:05Z
dc.degree.departmentChemistry
dc.degree.disciplineMS-Chemistry
dc.degree.grantorEast Carolina University
dc.degree.levelMasters
dc.degree.nameM.S.
dc.description.abstractProteins are dynamic in nature, with these motions playing a role in substrate binding and product release. Protein thermal motions have emerged as participating in the bond making/breaking steps of catalysis and by extension the rate enhancement observed in enzymes. A family of enzymes, known as lipoxygenases (LOXs), play a large role in growth and pathogenic defense in plants and homeostasis, cell signaling, and inflammation in humans. The regulation of LOX pro- and anti-inflammatory properties is thought to be controlled through allosteric interactions with small molecules, proteins, and membranes. For all organisms, LOXs oxidize polyunsaturated fatty acids through an often rate-limiting C-H activation step that proceeds through a tunneling mechanism. The activation energy barrier for this LOX reaction is expected to be related to the thermal fluctuations of the protein-substrate complex. How protein motions transfer heat from the surface to buried active sites remains an open question. Furthermore, the connection between thermal motions mediating allostery and the chemical step(s) are not well resolved. Recent studies on the model plant LOX, soybean lipoxygenase (SLO), have identified a solvent-exposed loop that is linked to the origins of a defined network for thermal activation that is distinct from the defined allosteric network. The human counterpart, human epithelial 15-lipoxygenase-2 (15-LOX-2), exhibits similar function but lacks some of these structural features found in SLO, thereby raising the question as to the evolution of structure and protein motions in these enzymes. In this thesis, biophysical methods, including temperature-dependent hydrogen deuterium exchange-mass spectrometry, X-ray crystallography, and differential scanning calorimetry, as well as enzyme kinetics are used to regionally define catalytically linked dynamics related to both allostery and chemical bond breaking step(s) of15-LOX-2 to further understand how thermal motions regulate lipoxygenase function.
dc.embargo.lift2024-05-01
dc.format.mimetypeapplication/pdf
dc.identifier.urihttp://hdl.handle.net/10342/10682
dc.language.isoen
dc.publisherEast Carolina University
dc.subjectprotein motions
dc.subjectallosteric regulation
dc.subjectthermal activation
dc.subjecthydrogen deuterium exchange-mass spectroscopy
dc.subject.lcshLipoxygenases
dc.subject.lcshEnzymology
dc.subject.lcshProteins--Conformation
dc.titleDefining Protein Motions the Comprise the Reaction Barrier in Human Epithelial 15-Lipoxygenase-2
dc.typeMaster's Thesis
dc.type.materialtext

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