Investigating the Rate-limiting Step of Indole-3-glycerol Phosphate Synthase
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
M. tuberculosis (Mt) is the bacterium that causes one of the oldest known diseases, tuberculosis (TB). The efficacy of first line antituberculosis drugs has diminished due to the increasing presence of multi-drug resistant strains of Mt. The enzyme indole-3-glycerol phosphate synthase (IGPS) in Mt (MtIGPS) was proposed to be a potential drug target due to its role in catalyzing the fourth step of the tryptophan biosynthetic pathway, a pathway essential for the survival of Mt. Investigating the rate-limiting step of the 3-step MtIGPS mechanism, including substrate binding, the chemical conversion of substrate to product, and product release, is essential in identifying a specific target for an inhibitor. For this purpose, wildtype MtIGPS, and single-point mutations E57N, E57D, and E168D were expressed and purified, and rate-determining experiments were conducted, in which D2O or glycerol were varied. A solvent deuterium kinetic isotope effect (SDKIE) experiment for wildtype revealed a 2-fold decrease in activity in the presence of D2O. E57N, E57D, and E168 mutations showed highest activity with increasing D2O, an inverse SDKIE. Single turnover experiments, in the presence of D2O, supported results of the SDKIE experiments. The SDKIE of wildtype MtIGPS indicates a proton transfer, in the conversion of substrate to product, is rate-limiting while the inverse SDKIE of MtIGPS mutations is indicative of a rate-determining step other than the proton transfer. This data provided an enhanced understanding of MtIGPS catalysis for the purpose of rational drug discovery.
Investigating the Rate-limiting Step of Indole-3-glycerol Phosphate Synthase
M. tuberculosis (Mt) is the bacterium that causes one of the oldest known diseases, tuberculosis (TB). The efficacy of first line antituberculosis drugs has diminished due to the increasing presence of multi-drug resistant strains of Mt. The enzyme indole-3-glycerol phosphate synthase (IGPS) in Mt (MtIGPS) was proposed to be a potential drug target due to its role in catalyzing the fourth step of the tryptophan biosynthetic pathway, a pathway essential for the survival of Mt. Investigating the rate-limiting step of the 3-step MtIGPS mechanism, including substrate binding, the chemical conversion of substrate to product, and product release, is essential in identifying a specific target for an inhibitor. For this purpose, wildtype MtIGPS, and single-point mutations E57N, E57D, and E168D were expressed and purified, and rate-determining experiments were conducted, in which D2O or glycerol were varied. A solvent deuterium kinetic isotope effect (SDKIE) experiment for wildtype revealed a 2-fold decrease in activity in the presence of D2O. E57N, E57D, and E168 mutations showed highest activity with increasing D2O, an inverse SDKIE. Single turnover experiments, in the presence of D2O, supported results of the SDKIE experiments. The SDKIE of wildtype MtIGPS indicates a proton transfer, in the conversion of substrate to product, is rate-limiting while the inverse SDKIE of MtIGPS mutations is indicative of a rate-determining step other than the proton transfer. This data provided an enhanced understanding of MtIGPS catalysis for the purpose of rational drug discovery.