Interaction between Citrate Synthase and Malate Dehydrogenase

Abstract

The interactions between pig heart citrate synthase and mitochondrial malate dehydrogenase or cytosolic malate dehydrogenase were studied using the frontal analysis method of gel filtration and by precipitation in polyethylene glycol. This method showed that an interaction between citrate synthase and mitochondrial malate dehydrogenase occurred but no interaction between citrate synthase and cytosolic malate dehydrogenase. Channeling of oxaloacetate in the malate dehydrogenase and citrate synthase-coupled systems was tested using polyethylene glycol precipitates of citrate synthase and mitochondrial malate dehydrogenase, and citrate synthase and cytosolic malate dehydrogenase. The effectiveness of large amounts of aspartate aminotransferase and oxaloacetate decarboxylase, as competing enzymes for the intermediate oxaloacetate, was examined. Aspartate aminotransferase and oxaloacetate decarboxylase were less effective competitors for oxaloacetate when precipitated citrate synthase and mitochondrial malate dehydrogenase in polyethylene glycol was used at low ionic strength compared with free enzymes in the absence of polyethylene glycol or with a co-precipitate of citrate synthase and cytosolic malate dehydrogenase. Substrate channeling of oxaloacetate with citrate synthase-mitochondrial malate dehydrogenase precipitate was inefficient at high ionic strength. These effects could be explained through electrostatic interactions of mitochondrial but not cytosolic malate dehydrogenase with citrate synthase.In the last twenty years it has been demonstrated that sequential enzymes which operate within a metabolic pathway interact with each other to form highly organized complexes (1, 2). The term metabolon was introduced to describe such enzyme-enzyme complexes (3, 4). Among the mitochondrial enzymes of the Krebs tricarboxylic acid cycle, interactions occur between six of eight sequential enzymes: fumarase with mitochondrial malate dehydrogenase (mMDH) 1 (5), mMDH with citrate synthase (CS) (6 -11), succinyl-CoA synthetase with ␣-ketoglutarate dehydrogenase complex (12), mitochondrial aconitase (mACO) with CS (13), NAD-dependent isocitrate dehydrogenase with mACO (14), and NAD-dependent isocitrate dehydrogenase with ␣-ketoglutarate dehydrogenase complex (15). Moreover, it has been shown that all of these enzymes bind to the inner surface of the mitochondrial inner membrane, whereas purified isozymes from other cellular compartments do not possess such binding abilities (13). Organized enzyme complexes may have several kinetic advantages for the cell (16,17). One possible advantage is preventing intermediates from escaping into solution where they may be sequestered by other enzymes for use in different metabolic pathways. In other words, the close proximity of consecutive enzymes of a metabolic pathway may be used to increase the metabolic flow through this pathway, especially by channeling the substrate at reduced bulk substrate concentration.However, there are methodological problems in demonstrating the in vitro exis...

Mitochondria from the left heart ventricles of both normotensive and spontaneously hypertensive rats oxidize externally added NADH mostly via a novel malate/oxaloacetate shuttle as reconstructed in vitro

“…In a control experiment, the occurrence of OAA outside mitochondria was confirmed in the supernatants of mitochondria previously added with malate alone. This was performed using both citrate synthase and malate dehydrogenase assays (30,31). In agreement with a previously published study (28), NADH oxidation was strongly inhibited (~85%) by 10 mM phenylsuccinate.…”

Reverse flux through cardiac NADP+-isocitrate dehydrogenase under normoxia and ischemia

“…Enzyme activities were assayed by monitoring the kinetics of optical density of NADH or NADPH (340 nm) measured on a Hewlett-Packard 8452A spectrophotometer. Citrate synthase and NAD ϩ -ICDH were assayed by standard procedures (22). The procedure of Nulton-Persson and Szweda (25) was slightly modified for aconitase.…”

Physical and Genetic Interactions of Cytosolic Malate Dehydrogenase with Other Gluconeogenic Enzymes

“…However, there is abundant experimental evidence for interactions between the mitochondrial tricarboxylic acid cycle isozyme of malate dehydrogenase and citrate synthase. This evidence has been obtained using a variety of methods including gel filtration chromatography and polyethylene glycol precipitation (17). In addition, kinetic models, based on yeast or mammalian fusion proteins containing citrate synthase fused to the amino terminus of malate dehydrogenase, have been presented to support the potential for channeling of oxaloacetate between the soluble enzymes (17,18,42).…”

“…This evidence has been obtained using a variety of methods including gel filtration chromatography and polyethylene glycol precipitation (17). In addition, kinetic models, based on yeast or mammalian fusion proteins containing citrate synthase fused to the amino terminus of malate dehydrogenase, have been presented to support the potential for channeling of oxaloacetate between the soluble enzymes (17,18,42). Vélot and Srere (19) also used a peptide "purtubagen," an in vivo competitor, to identify a region (residues 353-366) of yeast citrate synthase (CIT1) that appears to be important in functional interactions with MDH1 in vivo.…”