Abstract: Effects of MDH1 Mutations on Enzymatic Activity, Predicted Structure and pKa Values

Malate dehydrogenase (MDH) is an enzyme that catalyzes the reversible oxidation of malate to oxaloacetate as NAD+ is simultaneously reduced to NADH. There are several isozymes of malate dehydrogenase, but two main isoforms are found in eukaryotic cells (Minárik). One isoform is found in the mitochondrial matrix (MDH2) and acts as the key enzyme in the citric acid cycle that catalyzes the oxidation of malate. The other isoform (MDH1) is found in the cytoplasm and is known to assist the malate-aspartate shuttle, making it possible for malate to pass through the membrane and be used for other cellular processes (Musrati). This interconnection between MDH1 and other cellular pathways requires regulation of the enzyme. Phosphorylation is one regulatory mechanism, functioning to alter enzymatic activity. We have generated a mutated version of the recombinant enzyme at an amino acid that can be phosphorylated - the serine (S) located at amino acid 261 on MDH1- which is on the dimerization  interface. The region surrounding S261 is also divergent between the cytoplasmic and mitochondrial isoforms, and may play a key role in the different interactions of these enzymes. A mutation to aspartate (D), which is serine’s phosphomimetic, is likely to cause changes in MDH1’s structure and function, those of which are likely to disrupt enzymatic activity. An in vitro study of the enzyme kinetics of Wild Type MDH1 was originally planned, but we shifted our focus to an examination of predicted structural changes when S261 is mutated to aspartate, proline, glutamate, and lysine. We have examined MDH1 pKa values and structural changes through in silico experiments.