Investigating the Role of N189 in M.tuberculosis IGP Synthase and Experimental IC50 results of rCdRP and D-ribose Phosphate
Presentation Type
Poster
Faculty Advisor
Nina Goodey
Access Type
Event
Start Date
26-4-2024 12:45 PM
End Date
26-4-2024 1:44 PM
Description
Tuberculosis is one of the world’s leading infectious diseases. M. tuberculosis IGP Synthase (MtIGPS) catalyzes the conversion of substrate CdRP to IGP, which is an intermediate in the production of the essential amino acid tryptophan. We study MtIGPS to understand its function and to predict potential inhibitors that would interfere with its interaction with CdRP. I researched the mutated MtIGPS enzymes N189D and N189L, where the 189th amino acid was changed from a asparagine (N) to aspartic acid (D) and leucine (L), respectively. The N189D mutation changed the residue’s neutral amide charge to a negatively charged carboxyl group. The N189L mutation did not change the charge but changed the amide functional group in the sidechain to an isobutyl group. Compared to the wildtype, N189D and N189L reduced the catalytic activity of MtIGPS by 3,500-fold and 15,000-fold, respectively. N189D also weakened substrate binding and N189L had minimal impact on substrate binding. In addition, the compound D-ribose phosphate and the substrate analog rCdRP were tested as possible inhibitors of MtIGPS. It was determined that D-ribose phosphate did not inhibit MtIGPS up to a concentration of 15 mM. The substrate analog rCdRP did inhibit MtIGPS and an IC50 of 21 ± 2 µM was determined. I will continue to examine the mutants N189D, N189L, and other active site mutations, assessing steady state kinetics, pH profiles, and the rate limiting step of the original and mutated enzymes. I will also continue to test other compounds as potential inhibitors of MtIGPS.
Investigating the Role of N189 in M.tuberculosis IGP Synthase and Experimental IC50 results of rCdRP and D-ribose Phosphate
Tuberculosis is one of the world’s leading infectious diseases. M. tuberculosis IGP Synthase (MtIGPS) catalyzes the conversion of substrate CdRP to IGP, which is an intermediate in the production of the essential amino acid tryptophan. We study MtIGPS to understand its function and to predict potential inhibitors that would interfere with its interaction with CdRP. I researched the mutated MtIGPS enzymes N189D and N189L, where the 189th amino acid was changed from a asparagine (N) to aspartic acid (D) and leucine (L), respectively. The N189D mutation changed the residue’s neutral amide charge to a negatively charged carboxyl group. The N189L mutation did not change the charge but changed the amide functional group in the sidechain to an isobutyl group. Compared to the wildtype, N189D and N189L reduced the catalytic activity of MtIGPS by 3,500-fold and 15,000-fold, respectively. N189D also weakened substrate binding and N189L had minimal impact on substrate binding. In addition, the compound D-ribose phosphate and the substrate analog rCdRP were tested as possible inhibitors of MtIGPS. It was determined that D-ribose phosphate did not inhibit MtIGPS up to a concentration of 15 mM. The substrate analog rCdRP did inhibit MtIGPS and an IC50 of 21 ± 2 µM was determined. I will continue to examine the mutants N189D, N189L, and other active site mutations, assessing steady state kinetics, pH profiles, and the rate limiting step of the original and mutated enzymes. I will also continue to test other compounds as potential inhibitors of MtIGPS.