Date of Award
Master of Science (MS)
College of Science and Mathematics
Chemistry and Biochemistry
Thesis Sponsor/Dissertation Chair/Project Chair
Nina M. Goodey
Indole-3-glycerol phosphate synthesis (IGPS) is an enzyme that catalyzes the ring closure in 1-(o-carboxylphenylamino)-1-deoxyribulose 5-phosphate (CdRP). Multiple steps are expected to be involved in formation of the pyrrole ring including dehydration, decarboxylation, cyclization, and condensation. IGPS is an essential protein in the pathogen Mycobacterium tuberculosis and a potential target in the treatment of drug-resistant tuberculosis. In order to better understand the function of Mycobacterium tuberculosis IGPS (mtIGPS), we introduced single-point mutations into active site residues. This was based on the hypothesis that introducing mutations would affect the catalysis in a way where we would be able to obtain more information on our protein. We expressed and purified MtIGPS mutants N189L and E57D. The catalytic activities of the mutants were lower than that of the wild type mtIGPS, indicating that these residues play important roles in catalysis. The Michaelis constant, pH-dependence, and temperature dependence of the mutants were also determined. Wildtype mtIGPS was found to have a KM of 5.0 ± 1.4μM and a kcat value of 4.8 ± 0.3s-1 whereas E57D kinetics revealed a KM of 29 ± 6 μM and a kcat value of 0.102 ± 0.018s-1. N189L indicated no measurable activity and could not be further characterized. The data on E57D show an altered pH profile, possibly resulting from the lower pKa of Asp compared to Glu, and support the hypothesis that E57 serves as the catalytic base in mtIGPS mechanism. Together these data contribute to an improved understanding of the mtIGPS catalysis and ligand binding.
Cho, Sarah S., "Investigating the Effects of Catalytic Activity in Mycobacterium Tuberculosis Indole-3-Glycerol Phosphate Synthase in Wildtype, N189L, And E57D Variants" (2021). Theses, Dissertations and Culminating Projects. 753.
Available for download on Thursday, June 29, 2023