Biophysical Studies of Human Malic Enzymes

Tong

MCB9974700

Malic enzymes (ME) catalyze the oxidative decarboxylation of malate to produce pyruvate and CO2, with the concomitant reduction of the cofactor NAD or NADP. They are usually homo-tetramers, with molecular weights of about 60kD for the monomer. In mammals, three isoforms of malic enzymes have been identified so far - cytosolic NADP-dependent ME (c-NADP-ME), mitochondrial NADP-dependent ME (m-NADP-ME), and mitochondrial NAD (P)- dependent ME (m-NAD-ME). m-NAD-ME has several unique features among the three isoforms. It is a cooperative enzyme and exhibits sigmoidal kinetics with respect to the substrate malate. Its activity is allosterically controlled, with fumarate as an activator and ATP as an inhibitor. It can use both NAD and NADP as the co-factor, although it prefers NAD under physiological conditions. The structural basis for these unique properties are currently unknown. As a first step towards a greater understanding of these enzymes, the crystal structure of human m-NAD-ME in complex with NAD was determined recently. The structure demonstrates that malic enzymes are a new class of oxidative decarboxylases. This first structure represents the starting point for further biochemical, structural, and biophysical studies to characterize this important class of enzymes. Special emphasis will be placed on defining the catalytic mechanisms of malic enzymes in general, and on delineating the structural basis for the unique allosteric properties of m-NAD-ME in particular. Another aspect of the research will focus on the structural basis for the co-factor selectivity of the malic enzymes. m-NAD-ME offers the unique opportunity of studying the binding of NAD and NADP to the same enzyme. Structures of the NADP-dependent isoforms will also be obtained to provide additional information for comparison. The results obtained from the structural analyses will be assessed by mutagenesis and kinetic studies.

Malic enzymes are widely distributed in nature, having been found in bacteria, yeast, fungi, plants, and animals. Their amino acid sequences are highly conserved; suggesting that malic enzymes may have important biological functions. The proposed research should lead to a greater understanding of the biochemical properties and biological functions of this important class of enzymes. The research should also provide additional insights on the structural basis for the allosteric control of enzyme activities and the catalytic mechanisms of dehydrogenases and decarboxylases in general.