Biophysical Investigation into the Protein Dynamics Governing the Allosteric Regulation of Plant and Animal 15-Lipoxygenases

Lipoxygenases (LOXs) are a family of enzymes found in plants, animals, fungi, and bacteria that catalyze the per-oxidation of polyunsaturated fatty acids. In plants, LOXs are involved in growth, development, and defense against pathogenic attacks. There are also multiple isoforms present in humans, which have contradictory roles in the body. Specifically, human 15-LOX isoforms, 15-LOX-1 and 15-LOX-2, are involved in both homeostasis and pro-inflammatory pathways. In order to selectively target the activity of these enzymes, research has turned to allosteric regulation, which is the focus of this Thesis. Previously, the allosteric regulation of a model plant 15-LOX, soybean lipoxygenase-1 (SLO), has been characterized using hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing that the addition of the allosteric effector, oleyl sulfate (OS), alters a specific region of the enzyme. Herein, we used a combination of thermodynamic and biophysical techniques such as isothermal titration calorimetry and differential scanning calorimetry to investigate the allosteric regulation of SLO by OS. We present data which supports that the allosteric regulation of SLO by OS does not induce oligomerization or large-scale conformational changes and that the allostery is dynamically driven. We also employed HDX-MS to study the dynamics of 15-LOX-1 compared to previously collected data of 15-LOX-2 to reveal structural differences between the two isozymes that may explain their altered catalytic behavior.

Department dc:contributor.department

Chemistry

Grantor dc:publisher

East Carolina University

Year dc:date.issued

2022

Author dc:creator

• Roberts, Daniella

Advisor dc:contributor.advisor

• Offenbacher, Adam R

• Allostery
• Protein Dynamics
• Steady-State Kinetics
• Hydrogen-Deuterium Exchange Mass Spectrometry

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/10342/10684

OAI identifier oai:identifier

oai:thescholarship.ecu.edu:10342/10684