Succinate from isocitrate

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).

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... 

Fig. 2. (A) The glyoxylate cycle as a bypass of the TCA cycle (after Komberg and Krebs, 1957). (B) The glyoxylate cycle as it functions in the glyoxysome, showing the production of succinate from 2 mol of acetyl-CoA. The five steps constituting the cycle are catalyzed by the following enzymes (1) citrate synthetase, (2) aconitase, (3) isocitrate lyase, (4) malate synthetase, (5) malate dehydrogenase.

Another important piece of the puzzle came from the work of Carl Martius and Franz Knoop, who showed that citric acid could be converted to isocitrate and then to a-ketoglutarate. This finding was significant because it was already known that a-ketoglutarate could be enzymatically oxidized to succinate. At this juncture, the pathway from citrate to oxaloacetate seemed to be as shown in Figure 20.3. Whereas the pathway made sense, the catalytic effect of succinate and the other dicarboxylic acids from Szent-Gyorgyi s studies remained a puzzle. 

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 4. The citrate cycle. There is complete oxidation of one molecule of acetyl-CoA for each turn of the cycle CH3COSC0A + 2O2 - 2CO2 + H2O + CoASH. The rate of the citrate cycle is determined by many factors including the ADP/ATP ratio, NAD7NADH ratio, and substrate concentrations. During muscle contraction, Ca is released from cellular stores (mainly the sarcoplasmic reticulum) and then taken up in part by the mitochondria (see Table 2). Ca " activates 2-oxoglutarate and isocitrate dehydrogenases (Brown, 1992). Succinate dehydrogenase may be effectively irreversible. Enzymes ...

Figure 6.11 Separation of carboxylic acids (10 mJW each) by suppressed conductivity capillary electrophoresis. Conditions capillary, 60 cm X 75 jum I.D. fused silica voltage, +24 kV detection, suppressed conductivity using 15 mJV sulfuric acid as regenerant. Peaks (ppm) 1, quinic (1.92) 2, benzoic (1.44) 3, lactic (0.90) 4, acetic (0.60) 5, phthalic (1.66) 6, formic (0.46) 7, succinic (1.18) 8, malic (1.34) 9, tartaric (1.50) 10, fumaric (1.16) 11, maleic (1.16) 12, malonic (1.04) 13, citric (1.92) 14, isocitric (1.92) 15, cis-aconitic (1.74) 16, oxalic (0.90). (Reprinted from Ref. 63 with permission.)...

Figure 4>11. Data obtained through use of Dowex-1-formate columns for the separation of succinate, malate, and isocitrate. [From T. G. Cooper and H. Beevers, /. Biol. Chem., 244 3507-3513 (1969).]...

COs to form oxalacetate which under anaerobic conditions is reduced to malate. The malate in turn may be converted to fumarate and succinate (Fig, 5). The last step in this series of reactions is blocked by malonate. The second pathway involves the aerobic condensation of pyruvate and oxalacetate followed by oxidation of the condensation product to form -ketoglutarate and succinate. Wood has proposed that the first condensation product of the aerobic tricarboxylic cycle is cfs-aconitic acid which is then converted to succinate by way of isocitric, oxalosuccinic, and a-ketoglutaric acids. The a-ketoglutarate is decarboxylated and oxidized to succinic acid. Isotopic a-ketoglutarate containing isotopic carbon only in the carboxyl group located a to the carbonyl would be expected to yield non-isotopic succinate after decarboxylation. This accounts for the absence of isotopic carbon in succinate isolated from malonate-poisoned liver after incubation with pyruvate and isotopic bicarbonate. 

Aconitase catalyzes the isomerization of citrate to isodtrate, isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate, and a-ketoglutarate dehydrogenase catalyzes the oxidative decarboxylation of a-keto-glutarate to succinyl-CoA. Succinyl-CoA and the remaining intermediates are the 4-carbon intermediates of the Krebs cycle. Succinyl thiokinase catalyzes the release of coenzyme A from succinyl-CoA and the production of GTP. Succinate dehydro-... 

Figure 14.4 Separation of organic acids (as anions) and inorganic anions in wine (reproduced with permission of Dionex). Conditions column, 25cm x 4mm i.d. and precolumn stationary phase, lonPac AS11-HC mobile phase, 1.5 ml min nonlinear gradient from 1 mM to 60mM NaOH and from 0 to 20% methanol temperature, 30°C conductivity detector after packed suppressor. Peaks 1 = lactate 2 = acetate 3 = formate 4 = pyruvate 5 = galacturonate 6 = chloride 7 = nitrate 8 = succinate 9 = malate 10 —tartrate 11—fumarate 12 —sulfate 13 = oxalate 14 = phosphate 15 —citrate 16 = isocitrate 17 = c/s-aconitate 18 = frans-aconitate.

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