Effects of E47D amino acid on the catalytic activity and substrate binding affinity in
Presentation Type
Abstract
Faculty Advisor
Nina Goodey
Access Type
Event
Start Date
25-4-2025 12:00 PM
End Date
25-4-2025 1:00 PM
Description
Thermotoga maritima indole-3-glycerol phosphate synthase (TmIGPS) is a monomeric thermostable enzyme. Indole-3-glycerol phosphate synthase (IGPS) from Thermotoga maritima catalyzes the fifth step in the tryptophan biosynthesis pathway which function in the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate (CdRP) to indole-3-glycerol phosphate (IGP). This research investigates the temperature-dependent catalytic activity and substrate binding of a thermophilic enzyme TmIGPS, to understand its unique adaptations to environments such as hot springs and as a potential novel target. Enzyme kinetic assays, Kinetic solvent viscosity effects (KSVE) and Arrhenius plots were used to analyze the catalytic activity and substrate binding of wildtype and E47D TmIGPS at temperatures ranging from 25 °C to 80 °C. We proposed that the kinetic parameters governing these processes have different temperature dependencies. Previous studies reported that TmIGPS have a kcat value of 0.11 s-1 and Km value of 0.006 µM at 25 °C. Here we report the wildtype TmIGPS have a kcat value of 0.08 ± 0.002 s-1 and Km value of 0.057 ± 0.008 µM which suggests a 9.5-fold increase in substrate binding affinity. However, KSVE experiments revealed high catalytic activity at higher temperatures. E47D TmIGPS resulted in an increased Km value of 0.2 ± 0.06 µM at 25 °C, indicating lower substrate affinity. These results demonstrate that E47D mutation in the active site residues of IGPS drastically reduce its catalytic activity but contribute significantly to its thermostability. These findings into the mechanism of TmIGPS highlights its potential as an inhibitor.
Effects of E47D amino acid on the catalytic activity and substrate binding affinity in
Thermotoga maritima indole-3-glycerol phosphate synthase (TmIGPS) is a monomeric thermostable enzyme. Indole-3-glycerol phosphate synthase (IGPS) from Thermotoga maritima catalyzes the fifth step in the tryptophan biosynthesis pathway which function in the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate (CdRP) to indole-3-glycerol phosphate (IGP). This research investigates the temperature-dependent catalytic activity and substrate binding of a thermophilic enzyme TmIGPS, to understand its unique adaptations to environments such as hot springs and as a potential novel target. Enzyme kinetic assays, Kinetic solvent viscosity effects (KSVE) and Arrhenius plots were used to analyze the catalytic activity and substrate binding of wildtype and E47D TmIGPS at temperatures ranging from 25 °C to 80 °C. We proposed that the kinetic parameters governing these processes have different temperature dependencies. Previous studies reported that TmIGPS have a kcat value of 0.11 s-1 and Km value of 0.006 µM at 25 °C. Here we report the wildtype TmIGPS have a kcat value of 0.08 ± 0.002 s-1 and Km value of 0.057 ± 0.008 µM which suggests a 9.5-fold increase in substrate binding affinity. However, KSVE experiments revealed high catalytic activity at higher temperatures. E47D TmIGPS resulted in an increased Km value of 0.2 ± 0.06 µM at 25 °C, indicating lower substrate affinity. These results demonstrate that E47D mutation in the active site residues of IGPS drastically reduce its catalytic activity but contribute significantly to its thermostability. These findings into the mechanism of TmIGPS highlights its potential as an inhibitor.
Comments
Poster presentation at the 2025 Student Research Symposium.