Date of Award
8-2023
Document Type
Thesis
Degree Name
Master of Science (MS)
College/School
College of Science and Mathematics
Department/Program
Chemistry and Biochemistry
Thesis Sponsor/Dissertation Chair/Project Chair
Nina M. Goodey
Committee Member
David Konas
Committee Member
Quinn Vega
Abstract
Tuberculosis (TB) is a serious infectious disease caused by Mycobacterium tuberculosis. Tuberculosis drug resistance is increasingly a problem, but recent research has provided results that suggest Indole-3-Glycerol Phosphate Synthase (IGPS) could be a potential drug target. IGPS is an enzyme that catalyzes the fourth step in the tryptophan biosynthesis pathway, a crucial process for optimal Mycobacterium tuberculosis growth. In this research, we studied IGPS from Mycobacterium tuberculosis (MtIGPS) to shed light on a particular conserved residue that has not been studied in depth in any IGPS homolog. My investigation focuses on understanding the role of the active site residue E219. Through mutagenesis, I studied E219D, E219N, and E219Q to gain an understanding of what role it partakes in MtIGPS catalysis. We determined the wildtype in MtIGPS to have a kcat of 0.022 s-1 and when mutated caused significant shifts in catalytic activity. For instance; it was observed a 10.5-fold increase in catalytic activity was observed in E219Q. The mutant E219N and E219D showed a 2-fold and 81-fold reduction in catalytic activity, respectively. Through this investigation, we determined the length and charge in E219 are critical for MtIGPS catalysis. The steady-state kinetic and rate-pH profile’s results lead to the hypothesized role in E219 to form an important intermolecular interaction with the catalytic acid’s side chain K119. Therefore, orienting the catalytic acid K119 to proceed in its catalytic role in MtIGPS. Moreover, the solvent deuterium kinetic isotope effect revealed the wildtype’s rate-limiting step is an exchangeable proton transfer, but when E219 is mutated we observe the rate-limiting step to be of another step in the MtIGPS mechanism. Preliminary results at lower temperatures suggest the rate-limiting step can be altered depending on temperature. Furthermore, single-turnover experiments are intriguing as the chemical step is comparable to the kcat from steady-state kinetic results. Overall, results from this investigation give insight into a more detailed MtIGPS mechanism, especially in active site residue E219.
File Format
Recommended Citation
Moran, Cinthya, "Investigating the Effects of E219 Mutations in M. tuberculosis Indole-3-Glycerol Phosphate Synthase" (2023). Theses, Dissertations and Culminating Projects. 1356.
https://digitalcommons.montclair.edu/etd/1356