Malate from aspartate

To give a real example, have a closer look on main functions and cycle of magnesium in green plants. Control on autocatalysis depends on the principal functions of Mg, that is, on photosynthesis when substantial parts of Mg taken up by roots are allocated to chlorophyll and rubisco synthesis, less will be available for other metabolic pathways, reducing the turnovers there unless there are lots of Mg around like in marine plants. In addition, the tricarboxylate cycle (citrate cycle) requires Mg (besides Fe and Mn) to produce the enzymes hence some Mg (as well as Fe, Mn) must be invested to produce the citrate (malate, oxaloacetate (aspartate)) ions delivered by the roots to render Mg (and other metals) in turn bioavailable by means of complexation and resorption of almost neutral complex entities. Furthermore, the tricarboxylate cycle is coupled to biosynthesis of amino acids by redox transamination hence there will be both competition at the metal center(s) and possible extraction of metal ions from enzymes once NHj and electrons are... 

Electron shuttles Enzymatic processes whereby electrons from NADH can be transferred across the mitochondrial barrier. The glycerol 3-phosphate shuttle uses the reduction of dihydroxyacetone phosphate to glycerol 3-phosphate and reoxidation to transfer electrons from cytosolic NADH to coenzyme Q in the electron transport chain. The malate-aspartate shuttle uses malate and aspartate in a two-member transfer exchange to transfer electrons from cytosolic NADH to mitochondrial NADH (see Figures 27-2 and 27-3). 

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. 

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.

Oxaloacetate, produced from malate or aspartate in the cytosol, is converted to PEP by the cytosolic PEPCK (see Eig. 31.7A). 

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... 

C4 plants green plants in which the primary product of CO2 fixation is not 3-phosphoglycerate (cf. C3 plants) but a C4 acid such as oxaloacetate, malate or aspartate. These plants possess two types of photi>-synthesizing cells. In mesophyll cells near the leaf surface, CO2 is fixed into C4-compounds. This prefixation of CO2 is due to the action of the cytosolic enzyme, phosphoeno/pyruvate carboxylase (EC 4.1.1.31), which carboxylates phosphoenolpyruvate to oxaloacetic acid (see Hatch-Slack-Kortschak cycle). The Calvin cycle (see) operates in the the vascular bundle cells of C4 plants, and CO2 for the Calvin cycle is derived from the decarboxylation C4 compounds rather than directly from the atmosphere. This Kranz anatomy , i.e. photosynthetically active bundle sheath cells with a photosynthetically active layer... 

Two separation procedures were used to identify all products that could be formed from [4-aspartate. With the HVPE the end-product, OA, could be separated from the substrate and all other products, including citric acid cycle intermediates. Malate and fuma-rate had the same mobility as carbamyl aspartate and DHO, respectively, but could be separated by TLC. The identity of OA was confirmed by conversion of eluted radioactivity with partially purified yeast OPRT and ODC to OMP and UMP. With brain cortex the rate of OA synthesis from aspartate was 52 12 and with liver 179 35 nmol/h per g wet tissue (means SD of 7 and 4 experiments, respectively) expressed per mg protein these values were 0.81 0.21 and 1.12 0.46, respectively. With both tissues about 10% of the label was found in citric acid cycle intermediates, and with cortex and liver about 1% and 10% of radioactivity was recovered as 002 ... 

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. 

Unlike glycolysis, which occurs strictly in the cell cytosol, gluconeogen-esis involves a complex interaction between the mitochondrion and the cytosol. This interaction is necessitated by the irreversibility of the pyruvate kinase reaction, by the relative impermeability of the inner mitochondrial membrane to oxaloacetate, and by the specific mitochondrial location of pyruvate carboxylase. Compartmentation within the cell has led to the distribution of a number of enzymes (aspartate and alanine aminotransferases, and NAD -malate dehydrogenase) in both the mitochondria and the cytosol. In the classical situation represented by the rat, mouse, or hamster hepatocyte, the indirect "translocation" of oxaloacetate—the product of the pyruvate carboxylase reaction—into the cytosol is effected by the concerted action of these enzymes. Within the mitochondria oxaloacetate is converted either to malate or aspartate, or both. Following the exit of these metabolites from the mitochondria, oxaloacetate is regenerated by essentially similar reactions in the cytosol and is subsequently decarboxylated to P-enolpyruvate by P-enol-pyruvate carboxykinase. Thus the presence of a membrane barrier to oxaloacetate leads to the functioning of the malate-aspartate shuttle as an important element in gluconeogenesis. 

Glyoxysomes do not contain all the enzymes needed to run the glyoxylate cycle succinate dehydrogenase, fumarase, and malate dehydrogenase are absent. Consequently, glyoxysomes must cooperate with mitochondria to run their cycle (Figure 20.31). Succinate travels from the glyoxysomes to the mitochondria, where it is converted to oxaloacetate. Transamination to aspartate follows... 

Because the 2 NADH formed in glycolysis are transported by the glycerol phosphate shuttle in this case, they each yield only 1.5 ATP, as already described. On the other hand, if these 2 NADH take part in the malate-aspartate shuttle, each yields 2.5 ATP, giving a total (in this case) of 32 ATP formed per glucose oxidized. Most of the ATP—26 out of 30 or 28 out of 32—is produced by oxidative phosphorylation only 4 ATP molecules result from direct synthesis during glycolysis and the TCA cycle. 

Oxidation of 2 molecules each of isocitrate, n-ketoglutarate, and malate yields 6 NADH Oxidation of 2 molecules of succinate yields 2 [FADHg] Oxidative phosphorylation (mitochondria) 2 NADH from glycolysis yield 1.5 ATP each if NADH is oxidized by glycerol-phosphate shuttle 2.5 ATP by malate-aspartate shuttle + 3 + 5... 

Figure 12-13. Malate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion. Ketoglutarate transporter , glutamate/aspartate transporter (note the proton symport with glutamate).

Figure 9 A synthetic mixture of water-soluble carboxylic acids separated by anion-exchange chromatography. Column 0.3 cm x 300 cm Diaoion CA 08, 16-20 p (Mitsubishi Kasei Kogyo). Eluant 200 mM HC1. Detection reaction with Fe3-benzohy-droxamic acid-dicyclohexy carbodiimide-hydroxylamine perchlorate-triethyl amine with absorbance at 536 nm. Analytes (1) aspartate, (2) gluconate, (3) glucuronate, (4) pyroglutamate, (5) lactate, (6) acetate, (7) tartrate, (8) malate, (9) citrate, (10) succinate, (11) isocitrate, (12) w-butyrate, (13) a-ketoglutarate. (Reprinted with permission from Kasai, Y., Tanimura, T., and Tamura, Z., Anal. Chem., 49, 655, 1977. 1977 Analytical Chemistry).

Electrons from NADH outside the mitochondria are transported into the mitochondria by the malate-aspartate shuttle or the a-glycerol phosphate shuttle. 

The MALATE-ASPARTATE SHUTTLE gets reducing equivalents (electrons) from cytosolic NADH into the mitochondria so that 3 ATPs can be made. 

The malate-aspartate shuttle is the most important pathway for transferring reducing equivalents from the cytosol to the mitochondria in brain 541... 

The malate-aspartate shuttle is the most important pathway for transferring reducing equivalents from the cytosol to the mitochondria in brain. This shuttle involves both the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase, the mitochondrial aspartate-glutamate carrier and the dicarboxylic acid carrier in brain (Fig. 31-5) [69]. The electrogenic exchange of aspartate for glutamate and a... 

The fumarate produced in step [4] is converted via malate to oxaloacetate [6, 7], from which aspartate is formed again by transamination [9]. The glutamate required for reaction [9] is derived from the glutamate dehydrogenase reaction [8], which fixes the second NH4 " in an organic bond. Reactions [6] and [7] also occur in the tricarboxylic acid cycle. However, in urea formation they take place in the cytoplasm, where the appropriate isoenzymes are available. 

In the malate shuttle (left)—which operates in the heart, liver, and kidneys, for example-oxaloacetic acid is reduced to malate by malate dehydrogenase (MDH, [2a]) with the help of NADH+HT In antiport for 2-oxogluta-rate, malate is transferred to the matrix, where the mitochondrial isoenzyme for MDH [2b] regenerates oxaloacetic acid and NADH+HT The latter is reoxidized by complex I of the respiratory chain, while oxaloacetic acid, for which a transporter is not available in the inner membrane, is first transaminated to aspartate by aspartate aminotransferase (AST, [3a]). Aspartate leaves the matrix again, and in the cytoplasm once again supplies oxalo-acetate for step [2a] and glutamate for return transport into the matrix [3b]. On balance, only NADH+H"" is moved from the cytoplasm into the matrix ATP is not needed for this. 

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