Evaluating the Impact of K135T Mutation on Malate Dehydrogenase Kinetic Activity
Location
Oyate Hall
Event Website
https://2026undergraduateresearchsy.sched.com/event/2Ix8n/evaluating-the-impact-of-k135t-mutation-on-malate-dehydrogenase-kinetic-activity
Start Date
15-4-2026 6:00 PM
End Date
15-4-2026 8:00 PM
Description
Malate Dehydrogenase (MDH) is a key protein in the citric acid cycle; its dysregulation can have catastrophic impacts, such as neurological and metabolic disorders, and various cancers. MDH catalyzes the reversible oxidation-reduction reaction between malate and oxaloacetate, also reducing NAD+ to NADH in the process. The citric acid cycle is one of the central metabolic pathways, connecting to other essential pathways, such as amino acid and fatty acid biosynthesis, highlighting the importance of understanding how MDH works. A key site in MDH is the flexible loop region (amino acids 119-137), responsible for substrate binding, catalysis, and orientation. The flexibility of the loop region, in particular, enables the substrate to position itself correctly at the active site via electrostatic interactions, a poorly understood process. Therefore, in this study, we investigated the loop region to better understand how amino acids interact with the substrate. We hypothesized that replacing a positively charged lysine-135 with an uncharged threonine would destabilize the ligand and lower the binding affinity in the active site of MDH. To test this, we purified the mutant protein using nickel affinity chromatography and characterized it by SDS-PAGE, a Bradford assay, and enzyme kinetics. Enzyme assays helped determine that our mutant exhibited decreased enzymatic activity compared to the wild-type, supporting the stabilizing role of lysine-135 in the flexible loop region of MDH.
Publication Date
2026
Evaluating the Impact of K135T Mutation on Malate Dehydrogenase Kinetic Activity
Oyate Hall
Malate Dehydrogenase (MDH) is a key protein in the citric acid cycle; its dysregulation can have catastrophic impacts, such as neurological and metabolic disorders, and various cancers. MDH catalyzes the reversible oxidation-reduction reaction between malate and oxaloacetate, also reducing NAD+ to NADH in the process. The citric acid cycle is one of the central metabolic pathways, connecting to other essential pathways, such as amino acid and fatty acid biosynthesis, highlighting the importance of understanding how MDH works. A key site in MDH is the flexible loop region (amino acids 119-137), responsible for substrate binding, catalysis, and orientation. The flexibility of the loop region, in particular, enables the substrate to position itself correctly at the active site via electrostatic interactions, a poorly understood process. Therefore, in this study, we investigated the loop region to better understand how amino acids interact with the substrate. We hypothesized that replacing a positively charged lysine-135 with an uncharged threonine would destabilize the ligand and lower the binding affinity in the active site of MDH. To test this, we purified the mutant protein using nickel affinity chromatography and characterized it by SDS-PAGE, a Bradford assay, and enzyme kinetics. Enzyme assays helped determine that our mutant exhibited decreased enzymatic activity compared to the wild-type, supporting the stabilizing role of lysine-135 in the flexible loop region of MDH.
https://digitalcommons.morris.umn.edu/urs_event/2026/posters/7