Date of Award
5-2024
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 Goodey
Committee Member
David Konas
Committee Member
Jaclyn Catalano
Abstract
Tuberculosis (TB) remains a leading cause of adult mortality globally, with increasing drug resistance posing a significant challenge. Mycobacterium tuberculosis (Mt) is the causative agent, and understanding its enzymatic processes is crucial for drug development. One potential target is the enzyme Indole-3-glycerol phosphate synthase (IGPS), which plays a role in the tryptophan biosynthesis pathway and catalyzes the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose5’-phosphate (CdRP) into indole-3-glycerol phosphate (IGP). Investigating MtIGPS active site residues, particularly E57 and E219, through single- and double-point mutations revealed insights into their roles. Previous results of steady-state kinetics of the single-point mutations of E57D, E57Q, E219D, and E219Q indicated a substantial decrease in catalytic activity for E57D, E57Q, and E219D, and a 10-fold increase in catalytic activity for E219Q. The steady-state kinetics and rate-pH profiles of the double-point mutations E57D/E219Q and E57Q/E219Q were examined. Steady-state kinetics yielded a 200-fold and 1000-fold reduction in catalytic activity upon mutating the wild type to E57D/E219Q and E57Q/E219Q variants, respectively. Temperature-dependent studies on wildtype MtIGPS have demonstrated a significant increase in catalytic activity with rising temperature. A solvent deuterium kinetic isotope effect was conducted and found present in wildtype, indicating a proton transfer as the rate-limiting step at lower temperatures (17 °C – 34 °C), with a change in rate-limiting step at higher temperatures (37 °C – 46 °C). In contrast, E57D showed no solvent deuterium kinetic isotope effect at the same temperatures, indicating the rate-limiting step is not proton transfer. This data highlighted indicates the catalytic importance of residue E57 in CdRP-MtIGPS interactions.
File Format
Recommended Citation
Ezichi, Uchenna Emmanuel, "Investigating the Role of Residue E57 and Temperature Effects in Mycobacterium Tuberculosis Indole-3-Glycerol Phosphate Synthase Catalysis" (2024). Theses, Dissertations and Culminating Projects. 1425.
https://digitalcommons.montclair.edu/etd/1425