[
International Worm Meeting,
2011]
Malate dehydrogenase (MDH) is the last enzyme in the citric acid cycle. MDH catalyzes the conversion of NAD+ and malate to NADH and oxaloacetate. Since the forward reaction is energetically unfavorable, the reverse reaction is usually studied. Eukaryotes have two versions of this enzyme, one that is imported into the mitochondria and one that remains in the cytoplasm. The cytoplasmic MDH in C. elegans (F46E10.10) was originally mis-classified as lactate dehydrogenase. Hold and Riddle (Mech. Aging Dev. 124, 779, 2003) realized that the amino acids in the active site were compatible with malate binding rather than lactate. We named this enzyme MDH-1 and renamed the mitochondrial enzyme (F20H11.3) MDH-2 so that the naming convention corresponded to that used for other eukaryotes. We overexpressed MDH-1 in E. coli and removed the chitin binding domain tag used for the purification. In kinetic assays with the purified enzyme, MDH-1 had malate dehydrogenase activity that followed Michaelis-Menten kinetics. For the reverse reaction, we determined that the KM for oxaloacetate was 37 mM, and the KM for NADH was 70 mM. Our gel filtration results indicated that MDH-1 was active as a dimer, which is similar to the quaternary structure of most MDH enzymes (Minarik, P. et al., Gen. Physiol. Biophys. 21, 257, 2002). We are in the process of purifying endogenous MDH-1 from worms to compare its activity to our recombinant enzyme.