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

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Amanda Ohler (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Proteins 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) of 15-LOX-2 to further understand how thermal motions regulate lipoxygenase function.

Additional Information

Publication

Thesis

Language: English

Date: 2023

Subjects

lipoxygenase;enzymology;protein motions;allosteric regulation;thermal activation;hydrogen deuterium exchange-mass spectroscopy

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