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    Structure-Function Relationship of Surface N-glycans and Enzyme Active Site Proficiency: A Kinetic and Hydrogen-Deuterium Exchange Investigation of MoLOX

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    WHITTINGTON-MASTERSTHESIS-2021.pdf (2.843Mb)

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    Author
    Whittington, Chris
    Access
    This item will be available on: 2023-07-01
    Abstract
    A 'unique' enzyme has emerged within the lipoxygenase (LOX) family. This manganese containing enzyme comes from the pathogen Magnaporthe oryzae and termed MoLOX. MoLOX is secreted at the onset of pathogenesis when the rice blast fungus infects a rice plant that accounts for the loss of 1/3 of the world's rice crops. MoLOX has many different features when compared to the rest of the LOX family, including the presence of eight N-linked glycans at the surface of the enzyme, which is the focus of this Thesis. We used multiple methods to remove the glycans from the surface including treatment with PNGase F and Endo H as well as asparagine to glutamine site directed mutagenesis. Steady state kinetics was employed to determine the impact of glycan removal on first- and second-order kinetic parameters for protio substrates as well as using a deuterated substrate to help determine the impact on the kinetic isotope effects, useful probes in the case of LOX C-H activation. In addition, hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to determine the effect of glycan removal on regional protein flexibility. From these studies, we have identified regions of the protein that account for altered catalytic proficiency and substrate selectivity that depends upon the deglycosylation method deployed.
    URI
    http://hdl.handle.net/10342/9385
    Subject
     MoLOX; HDX; magnaporthe oryzae; LOX; N-glycans 
    Date
    2021-07-08
    Citation:
    APA:
    Whittington, Chris. (July 2021). Structure-Function Relationship of Surface N-glycans and Enzyme Active Site Proficiency: A Kinetic and Hydrogen-Deuterium Exchange Investigation of MoLOX (Master's Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/9385.)

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    MLA:
    Whittington, Chris. Structure-Function Relationship of Surface N-glycans and Enzyme Active Site Proficiency: A Kinetic and Hydrogen-Deuterium Exchange Investigation of MoLOX. Master's Thesis. East Carolina University, July 2021. The Scholarship. http://hdl.handle.net/10342/9385. August 12, 2022.
    Chicago:
    Whittington, Chris, “Structure-Function Relationship of Surface N-glycans and Enzyme Active Site Proficiency: A Kinetic and Hydrogen-Deuterium Exchange Investigation of MoLOX” (Master's Thesis., East Carolina University, July 2021).
    AMA:
    Whittington, Chris. Structure-Function Relationship of Surface N-glycans and Enzyme Active Site Proficiency: A Kinetic and Hydrogen-Deuterium Exchange Investigation of MoLOX [Master's Thesis]. Greenville, NC: East Carolina University; July 2021.
    Collections
    • Master's Theses
    Publisher
    East Carolina University

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