Abstract: Mutating MDH1 at Serine 264 and Its Impact on Dimerization and pKa

Malate dehydrogenase is an enzyme involved in cellular metabolism, which reversibly catalyzes the oxidation of malate to oxaloacetate using the reduction of NAD+ to NADH. Human cytosolic malate dehydrogenase (MDH1) is a critical enzyme in the malate-aspartate shuttle, which contributes to the transfer of cytosolic NADH into the mitochondria. Previous studies have found that when MDH1 is monomethylated on arginine 248, the post-translational modification prevents dimerization and inhibits catalytic activity. In order to further examine the role of post-translational modifications in MDH1 dimerization, we focused our study on the phosphorylation of serine 263, which is also found in the dimer interface. Mimicking phosphorylation on serine 263 to change the residue to an aspartic acid could possibly prevent dimerization of MDH1 in humans. Thus, this experiment examines the structure of cMDH and the role of the amino acid Ser263 to the dimerization of the enzyme. We generated an oligonucleotide mutation on the amino acid of the recombinant plasmid, and changed S263 to aspartate (D) in order to mimic phosphorylation. To observe potential effects in structure that other mutations could do, we processed three additional mutations (S263E,  S263F, and S263) through PyMOL, a cross-platform molecular graphics tool widely used for 3D visualization of proteins, and Phyre2, a web-based service for protein structure prediction. We also used H++, a system that provides a set of tools for analysis of electrostatic-related molecular properties, to analyze differences in pKa among the mutants. Results of these investigations are reported. Future in vitro experiments will be performed to determine the effect of the S263D mutant on enzyme kinetics parameters.