Rewiring an Enzyme: K135F Mutation in Malate Dehydrogenase

Malate Dehydrogenase (MDH) is an enzyme that catalyzes the reaction of malate to oxaloacetate by reducing the cofactor NAD+ to NADH in the citric acid cycle, playing a crucial role in cellular metabolism. Alterations in the MDH structure have been linked to metabolic diseases and cancers, though its overall structure remains poorly characterized. To better understand MDH, we manipulated its structure by inserting a point mutation into the flexible loop region at position 135, exchanging a lysine (Lys) to a phenylalanine (Phe). We hypothesized that mutating a positively charged amino acid (Lys) to an aromatic amino acid (Phe) would cause aromatic-aromatic binding with the phenylalanine in position 134. We predicted this structural change would decrease the flexibility of the loop region, slowing the enzymatic activity of MDH. Using PyMol modeling, we discovered that the Phe135 increased the distance between surrounding amino acids, likely disrupting these interactions. To test our findings, the mutated MDH protein was expressed in E. coli cells and purified by column chromatography. Purified mutated MDH was confirmed by an SDS-PAGE gel and Bradford assay. To study the effects of the mutation, an enzyme assay evaluating the velocity and binding of the protein to the substrate was performed. We found that the mutant enzyme had a low velocity and binding affinity, resulting in a 97.77% decrease in specific activity and turnover rate when compared to wild-type. These results confirm our hypothesis and give us insight into how the structure is important for functionality.