Proline to Serine Mutation in Flexible Loop Region of Malate Dehydrogenase
Location
Oyate Hall
Event Website
https://2026undergraduateresearchsy.sched.com/event/2Ix8e/proline-to-serine-mutation-in-flexible-loop-region-of-malate-dehydrogenase
Start Date
15-4-2026 6:00 PM
End Date
15-4-2026 8:00 PM
Description
Malate Dehydrogenase (MDH) is an essential enzyme that converts malate to oxaloacetate using the reduction of NAD+ to NADH. In humans, MDH plays a role in energy metabolism by catalyzing a key step of the citric acid cycle, important for energy production. Other indirect roles for MDH include gluconeogenesis and reoxidizing NADH from fatty-acid β-oxidation. An important feature of MDH is a flexible loop region that can alter substrate binding and enzyme activity, but its role is not well understood. Our study investigated how Pro123 to Ser (P123S) mutation in the flexible loop region affects MDH function. We hypothesized that the P123S mutation would increase loop mobility and improve substrate specificity as proline involves a secondary amine in its backbone and serine does not. To test this hypothesis, we performed site-directed mutagenesis, we expressed and purified His-tagged MDH in E.coli, and characterized the protein using Bradford assays, SDS-PAGE, and enzyme kinetic assays. The results revealed the P123S mutant protein showed lower specific activity, reduced turnover number, increased Km, and decreased Vmax. These results indicate that the mutation disrupts efficient catalysis, contrary to our initial hypothesis. Overall, this study shows the importance of P123 for proper loop structure and catalytic alignment for MDH function. It also provides insight into how mutations can impact proteins involved in enzyme function and metabolic pathways.
Publication Date
2026
Proline to Serine Mutation in Flexible Loop Region of Malate Dehydrogenase
Oyate Hall
Malate Dehydrogenase (MDH) is an essential enzyme that converts malate to oxaloacetate using the reduction of NAD+ to NADH. In humans, MDH plays a role in energy metabolism by catalyzing a key step of the citric acid cycle, important for energy production. Other indirect roles for MDH include gluconeogenesis and reoxidizing NADH from fatty-acid β-oxidation. An important feature of MDH is a flexible loop region that can alter substrate binding and enzyme activity, but its role is not well understood. Our study investigated how Pro123 to Ser (P123S) mutation in the flexible loop region affects MDH function. We hypothesized that the P123S mutation would increase loop mobility and improve substrate specificity as proline involves a secondary amine in its backbone and serine does not. To test this hypothesis, we performed site-directed mutagenesis, we expressed and purified His-tagged MDH in E.coli, and characterized the protein using Bradford assays, SDS-PAGE, and enzyme kinetic assays. The results revealed the P123S mutant protein showed lower specific activity, reduced turnover number, increased Km, and decreased Vmax. These results indicate that the mutation disrupts efficient catalysis, contrary to our initial hypothesis. Overall, this study shows the importance of P123 for proper loop structure and catalytic alignment for MDH function. It also provides insight into how mutations can impact proteins involved in enzyme function and metabolic pathways.
https://digitalcommons.morris.umn.edu/urs_event/2026/posters/5