Oxaloacetate conversion from malate

Answer C. Oxaloacetate, produced from pyruvate, exits the mitochondrion after conversion to malate. 

Acetic acid measurement involves conversion of acetate to acetyl-CoA by acetyl-CoA synthetase with the consumption of ATP (Boehringer Mannheim, 1986). Acetyl-CoA reacts with oxaloacetate and water in the presence of citrate synthetase to form citrate and CoA. Oxaloacetate for this reaction is obtained from malate by the action of malate dehydrogenase with concomitant conversion of NADH from NAD+. NADH is spectrophotometrically measured and correlated to acetic acid concentration. 

The answer is b. (Murray, pp 182-189. Scriver, pp 1521-1552. Sack, pp 121-138. Wilson, pp 287-317.) Reducing equivalents are produced at four sites in the citric acid cycle. NADH is produced by the isocitrate dehydrogenase-catalyzed conversion of a-ketoglutarate to succinyl CoA and by the malate dehydrogenase-catalyzed conversion of malate to oxaloacetate. FADH, is produced by the succinate dehydrogenase-catalyzed conversion of succinate to fumarate. Succinyl CoA synthetase catalyzes the formation of succinate from succinyl CoA, with the concomitant phosphorylation of GDP to GTP... 

Amino acids that form intermediates of the TCA cycle are converted to malate, which enters the cytosol and is converted to oxaloacetate, which proceeds through gluconeogenesis to form glucose. When excessive amounts of ethanol are ingested, elevated NADH levels inhibit the conversion of malate to oxaloacetate in the cytosol. Therefore, carbons from amino acids that form intermediates of the TCA cycle cannot be converted to glucose as readily. 

Oxaloacetate, generated from pyruvate by pyruvate carboxylase or from amino acids that form intermediates of the TCA cycle, does not readily cross the mitochondrial membrane. It is either decarboxylated to form PEP by the mitochondrial PEPCK or it is converted to malate or aspartate (see Figs. 31.7B and 31.7C). The conversion of oxaloacetate to malate requires NADH. PEP, malate, and aspartate can be transported into the cytosol. 

Although the major route for aspartate degradation involves its conversion to oxaloacetate, carbons from aspartate can form fumarate in the urea cycle (see Chapter 38). This reaction generates cytosolic fumarate, which must be converted to malate (using cytoplasmic fumarase) for transport into the mitochondria for oxidative or anaplerotic purposes. An analogous sequence of reactions occurs in the purine nucleotide cycle. Aspartate reacts with inosine monophosphate (IMP) to... 

A more complex and more efficient shutde mechanism is the malate-aspartate shuttle, which has been found in mammalian kidney, liver, and heart. This shuttle uses the fact that malate can cross the mitochondrial membrane, while oxaloacetate cannot. The noteworthy point about this shuttle mechanism is that the transfer of electrons from NADH in the cytosol produces NADH in the mitochondrion. In the cytosol, oxaloacetate is reduced to malate by the cytosolic malate dehydrogenase, accompanied by the oxidation of cytosolic NADH to NAD+ (Figure 20.24). The malate then crosses the mitochondrial membrane. In the mitochondrion, the conversion of malate back to oxaloacetate is catalyzed by the mitochondrial malate dehydrogenase (one of the enzymes of the citric acid cycle). Oxaloacetate is converted to aspartate, which can also cross the mitochondrial membrane. Aspartate is converted to oxaloacetate in the cytosol, completing the cycle of reactions. 

The conversion of malate to oxaloacetate has a AG° = +7.1 kcal/mol, yet in the citric acid cycle the reaction proceeds from malate to oxaloacetate. Explain how this is possible. 

But it was known that fumarase catalyses the malate-fiimarate transformation, and that malic dehydrogenase catalyses the oxaloacetate-malate conversion. From which facts sprung a new formulation of the cycle of the dicarboxylic acids. 

In discussion of nomenclature of malic acid decomposing enzymes, mention should be made of malate-lactate transhydrogenase. This enzyme, isolated from Micrococcus lactiyticus (VielloneUa alcalescens), a bacterium found in vertibrates, can catalyze reversibly the conversion of L-malic and pyruvic acids to L-lactic and oxaloacetic acids with NAD as coenzyme (36). 

Cycling between mitochondria and cytosol is the supply of acetyl-CoA for use in biosynthetic sequences in the cytosol. Citrate moves from the interior of the mitochondria to the cytosol. In the cytosol it is cleaved to acetyl-CoA and oxaloacetate by citrate lyase. The equilibrium constant for this reaction is favorable because an ATP-to-ADP conversion is involved. Most of the oxaloacetate is reduced to malate. The malate may be taken up by mitochondria or oxidized to pyruvate and C02, generating NADPH for use in biosynthetic sequences in the cytosol. The pyruvate enters the mitochondria, where it may be converted to oxaloacetate or acetyl-CoA by the usual routes (see fig. 13.14). 

Gluconeogenesis from pyruvate is not equal to the reverse process of glycolytic degradation of glucose to this 3-carbon intermediate. The glycolytic pathway and the gluconeogenetic pathway deviate at three steps. The conversion of pyruvate to PEP is not mediated by pyruvate kinase due to the irreversible nature of this metabolic step. Pyruvate, derived from either lactate or alanine, is converted to oxaloacetate in the mitochondrial matrix. This step is catalyzed by pyruvate carboxylase. Oxaloacetate per se cannot pass the mitochondrial inner membrane. However, with the use of the malate-aspartate shuttle, the 4-carbon skeleton of oxaloacetate can be transferred into the cytoplasmic compartment. Then oxaloacetate is converted to PEP by the action of PEP carboxykinase (Figure 1). 

When H, = H, H, = H and H = H when H, = H, H, = H and Hi, = H. The (35)-[3- H,2H]- and (3S)-[3-3H, H]oxaloacetates were enzymically synthesized from stereospecifically labeled samples of aspartate using glutamate-oxaloacetaie transaminase. Configurational analysis of the samples of [ H, H H] pyruvate involved enzymic conversion to chiral labeled acetate (lactate dehydrogenase/lactate oxidase), and the sequential use of malate synthase and fiimarase (see text) (223). 

Fig. 31.5. Conversion of pyruvate to phosphoenolpyruvate (PEP). Follow the shaded circled numbers on the diagram, starting with the precursors alanine and lactate. The first step is the conversion of alanine and lactate to pyruvate. Pyruvate then enters the mitochondria and is converted to OAA (circle 2) by pyruvate carboxylase. Pyruvate dehydrogenase has been inactivated by both the NADH and acetyl-CoA generated from fatty acid oxidation, which allows oxaloacetate production for gluconeogenesis. The oxaloacetate formed in the mitochondria is converted to either malate or aspartate to enter the cytoplasm via the malate/aspartate shuttle. Once in the cytoplasm the malate or aspartate is converted back into oxaloacetate (circle 3), and phosphoenolpyruvate carboxykinase will convert it to PEP (circle 4). The white circled numbers are alternate routes for exit of carbon from the mitochondrion using the malate/aspartate shuttle. OAA = oxaloacetate FA = fatty acid TG = triacylglycerol.

F. 31.7. The generation of PEP from gluconeogenic precursors. A. Conversion of oxaloacetate to phosphoenolpyruvate, using PEP carboxykinase. B. Interconversion of oxaloacetate and malate. C. Transamination of aspartate to form oxaloacetate. Note that the cytosolic reaction is the reverse of the mitochondrial reaction as shown in Eigure 31.5. 

Unlike most organisms, C. thermocellum does not encode a pyruvate kinase, which would generate 2 ATP during the conversion of 2 PEP to pyruvate. Instead, the reaction(s) responsible for conversion of PEP to pyruvate are uncertain (Figure 10.2d). One possibility is the so-called malate shunt [55, 57], in which PEP is first carboxylated to oxaloacetate (OAA) with the concomitant synthesis of GTP, which can be used directly to make ATP via nucleoside-diphosphate kinase. Then, OAA can either be directly decarboxylated to pyruvate or reduced to malate and then oxidatively decarboxylated to pyruvate. The net result of these pathways is the synthesis of 2 ATP equiv. per glucose, the same as using pyruvate kinase, with the possibility of electron transfer from NADH to make NADPH. 

The conversion of oxaloacetate to succinate is catalyzed by enzymes of the citric acid cycle malate dehydrogenase, fumarase and succinate dehydrogenase. These enzymes were isolated from the cells of P. shermanii... 

The irreversible step of glycolysis catalysed by pyruvate kinase is bypassed in gluconeogenesis by conversion of pyruvate first to oxaloacetate, then conversion of oxaloacetate to phosphoenolpyruvate by phosphoenolpyru-vate carboxykinase. The transfer of the amino group from glutamate to oxaloacetate produces aspartate, catalysed by the enzyme aspartate aminotransferase. Malate dehydrogenase converts oxaloacetate to malate in the malate-aspartate shutde. 

Oxaloacetate is an intermediate of many metabolic pathways. It also plays a role in the malate-aspartate shuttle, which transfers high energy electrons into mitochondria. Citrate is formed by the condensation of oxaloacetate with acetyl CoA. A transamination reaction transfers an amino group from an amino acid to an a-keto acid. Transfer of the amino group from aspartate to a-ketoglutarate forms oxaloacetate and glutamate. In gluconeogenesis, pyruvate is carboxylated in mitochondria to form oxaloacetate. After transfer to the cytosol, the enzyme phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate to phosphoenolpyruvate. 

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