Date of Award

1-2026

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 Rotella

Committee Member

Jaclyn Catalano

Abstract

Tuberculosis is an infectious disease that affects millions of people worldwide, and antibiotic resistance has become a growing concern in treatment. Mycobacterium tuberculosis is the bacterial pathogen responsible for tuberculosis. Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fourth step of the tryptophan biosynthesis pathway. It has been suggested as a novel drug target in treating tuberculosis, as it is essential for survival. It catalyzes the conversion of the substrate, 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) to the product, indole-3-glycerol phosphate (IGP). Understanding the structural and functional roles of active site amino acid residues is essential for developing a potential inhibitor of IGPS. The Ser220 residue has been hypothesized as an important amino acid residue in the active site, as it forms a hydrogen bond with CdRP. To determine the significance of Ser220 residue, Ser220 was mutated to a threonine (Ser220Thr) and was expressed and purified. Michaelis-Menten kinetics were performed to determine the kinetic parameters, k꜀ₐₜ and Kₘ, of Ser220Thr. Single turnover kinetics were also used to determine the kₒbₛ values under different conditions to determine the rate-limiting step. The solvent deuterium kinetic isotope effect was evaluated, and produced similar rates as H₂O PIPES, indicating an absence of a proton transfer event being rate-limiting. Solvent viscosity effects in 10% glycerol were performed and displayed similar kₒbₛ values as the H₂O and D₂O PIPES and ruled out conformational changes being rate-limiting. An enzyme concentration dependence on rate was noted, as a linear relationship was observed between enzyme concentration and kₒbₛ values. Kintek Explorer simulations performed also provided insight into rate constants. Together, the observations from these experiments are consistent with a decarboxylation step being rate-limiting. Understanding the role of active site amino acid residues, such as Ser220, is essential in the development of a potential inhibitor.

File Format

PDF

Download

Available for download on Sunday, February 27, 2028

Share

COinS