Ph.D. (Doctor of Philosophy)
Department of Chemistry and Biochemistry
This dissertation focuses on method development for enzymatic activity and inhibition assays for use in screening potential antibacterial compounds. Infection by antibiotic-resistant pathogens in on the rise globally and several enzymes, including the those in the methylerythritol phosphate (MEP) pathway and fructose bisphosphate aldolase, as strong targets for novel pharmaceuticals because they are not present in humans. Several assays exist for the MEP pathway and aldolase, but there is always work to be done to improve sensitivity, increase throughput, and lower cost. This dissertation describes the development of several methods - hydrophilic interaction chromatography (HILIC), liquid chromatography-mass spectrometry (LC-MS), and high resolution-mass spectrometry (HR-MS) - to detect and quantitative enzymatically-relevant analytes. The HILIC method was used to identify nucleotide analytes from the MEP pathway and was applied to LC-MS with no post-column desalting. In addition, the procedure was transferred for the analysis of fructose bisphosphate (FBP), the substrate of the aldolase reaction, with no changes. Additionally, an HR-MS was developed and validated to detect and quantitate FBP with high specificity and precision. Furthermore, the validated HR-MS method was used to obtain kinetic data from rabbit muscle aldolase uninhibited and with known inhibitors adenosine monophosphate (AMP) and adenosine triphosphate (ATP). Results confirmed that the HR-MS method was able to detect and quantitate enzyme turnover in quenched aliquots of reaction mixture. Future work will focus on using HR-MS detection to continuously monitor reactions in situ in order to save time, labor, and cost. Finally, the goal is to combine the HILIC method with the continuous HR-MS method to study kinetics and inhibition of a variety of target enzymes against libraries of potential inhibitors in vitro and in vivo.
Carr, Allison Fabino, "Development of chromatography and mass spectrometry methods for enzyme kinetics and inhibition in glycolysis and the methylerythritol phosphate (MEP) pathways" (2019). Graduate Research Theses & Dissertations. 2187.
Northern Illinois University
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