Abstract: The Effects of Serine260 Phosphorylation on the Dimer Interface of Cytosolic Malate Dehydrogenase

Malate dehydrogenase (MDH) is a dimeric enzyme that catalyzes the reversible oxidation of malate to oxaloacetate through the reduction of NAD+ to NADH in various pathways, one being the malate-aspartate shuttle. The malate-aspartate shuttle utilizes the cytosolic form (MDH1) to allow for transport of malate across membranes. This study focuses on investigating MDH1 and the effects of phosphorylation of the serine 260 amino acid residue on the enzyme’s function. This residue is of interest due to its location in the enzyme dimer interface. It is known from other biochemical pathways that phosphorylation often serves a regulatory function and affects activity. In order to test this, site-directed mutagenesis was performed on serine 260 to replace it with a negative aspartate residue, which will act as a phosphomimetic, mirroring phosphorylation. Modeling software was utilized to study the effects of the phosphomimetic and two additional in silico mutations to see if the predicted 3D structure and the pKas of the side chains of the enzyme change. If changes in the characteristics are seen, then it is likely that phosphorylation and mutation of the site hinders dimerization. In future studies, this mutant will be used in in vitro assays, including enzyme activity assays, to investigate the effects of the mutation on the enzymatic parameters of the enzyme.