Investigating the Roles of Residue E57 and E219 and Temperature Effects on Mycobacteria Tuberculosis Indole 3 Glycerol Phosphate Synthase
Presentation Type
Poster
Faculty Advisor
Nina Goodey
Access Type
Event
Start Date
26-4-2024 9:45 AM
End Date
26-4-2024 10:44 AM
Description
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-deoxyribulose 5’-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 wildtype 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 residues E57 and E219 in MtIGPS.
Investigating the Roles of Residue E57 and E219 and Temperature Effects on Mycobacteria Tuberculosis Indole 3 Glycerol Phosphate Synthase
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-deoxyribulose 5’-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 wildtype 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 residues E57 and E219 in MtIGPS.