Abstract
Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P)-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P)-ME_R67A/R91A and m-NAD(P)-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P)-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P)-ME enzyme activity.
Highlights
Malic enzymes (MEs) are a family of Mg2+ or Mn2+-dependent oxidative decarboxylases that catalyze L-malate to CO2 and pyruvate, with a concomitant reduction of NAD(P)+ to NAD(P)H [1,2,3,4]
There are three isoforms distributed within the cells according to their subcellular localization and cofactor specificity: the cytosolic NADP+-dependent malic enzyme [5,6], the mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME, ME2) [2,7,8] and the mitochondrial NADP+-dependent malic enzyme (m-NADP-ME, ME3) [9]. c-NADP-ME is expressed in the liver and adipose tissues [1,5] and generates the NADPH required for the biosynthesis of long-chain fatty acids and steroids [1,5,7,9] m-NADP-ME is found in tissues with low division rates, such as heart, muscle and brain tissue, and it generates the NADPH for fatty acid biosynthesis [1]
C-NADP-ME is unresponsive to fumarate activation
Summary
Malic enzymes (MEs) are a family of Mg2+ or Mn2+-dependent oxidative decarboxylases that catalyze L-malate to CO2 and pyruvate, with a concomitant reduction of NAD(P)+ to NAD(P)H [1,2,3,4]. Various structures of human m-NAD(P)-ME, including an open form without substrate and metal ion and a closed form with a bound substrate analog and all cofactors and regulators, are available [12,22,23,24]. Earlier studies by our group demonstrated that some ionic amino acid residues that are not conserved among the different malic enzyme isoforms, including Lys, Glu, Lys and Asp102, have remarkable effects on fumarate-induced activation [12,26,27,28]. Doi:10.1371/journal.pone.0098385.t001 effect of structural analogs of the substrate malate and the allosteric activator fumarate on human m-NAD(P)-ME and, suggest that dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. Diethyl oxaloacetate was found to be an allosteric inhibitor of human mNAD(P)-ME
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