Citric acid cycle succinate formation

As a result of oxidations catalyzed by the dehydrogenases of the citric acid cycle, three molecules of NADH and one of FADHj are produced for each molecule of acetyl-CoA catabohzed in one mrn of the cycle. These reducing equivalents are transferred to the respiratory chain (Figure 16-2), where reoxidation of each NADH results in formation of 3 ATP and reoxidation of FADHj in formation of 2 ATP. In addition, 1 ATP (or GTP) is formed by substrate-level phosphorylation catalyzed by succinate thiokinase. 

Although the citric acid cycle directly generates only one ATP per turn (in the conversion of succinyl-CoA to succinate), the four oxidation steps in the cycle provide a large flow of electrons into the respiratory chain via NADH and FADH2 and thus lead to formation of a large number of ATP molecules during oxidative phosphorylation. 

Since in the citric acid cycle there is no net production of its intermediates, mechanisms must be available for their continual production. In the absence of a supply of oxalacetic acid, acctaic" cannot enter the cycle. Intermediates for the cycle can arise from the carinxylation of pyruvic acid with CO, (e.g., to form malic acid), the addition of CO > to phosphcnnlpyruvic acid to yield oxalacetic acid, the formation of succinic acid from propionic acid plus CO, and the conversion of glutamic acid and aspartic acid to alpha-ketoglutaric acid and oxalacetic acid, respectively. See Fig. 3. 

Three modifications of the conventional oxidative citric acid cycle are needed, which substitute irreversible enzyme steps. Succinate dehydrogenase is replaced by fumarate reductase, 2-oxoglutarate dehydrogenase by ferredoxin-dependent 2-oxoglutarate oxidoreductase (2-oxoglutarate synthase), and citrate synthase by ATP-citrate lyase [3, 16] it should be noted that the carboxylases of the cycle catalyze the reductive carboxylation reactions. There are variants of the ATP-driven cleavage of citrate as well as of isocitrate formation [7]. The reductive citric acid... 

Answer Because pyruvate carboxylase is a mitochondrial enzyme, the [14C]oxaloacetate (OAA) formed by this reaction mixes with the OAA pool of the citric acid cycle. A mixture of [1-14C] and [4-14C] OAA eventually forms by randomization of the C-l and C-4 positions in the reversible conversions OAA —> malate —> succinate. [1-14C] OAA leads to formation of [3,4-14C]glucose. 

The formation of acetyl-CoA from pyruvate in animals is via the pyruvate dehydrogenase complex, which catalyzes the irreversible decarboxylation reaction. Carbohydrate is synthesized from oxaloacetate, which in turn is synthesized from pyruvate via pyruvate carboxylase. Since the pyruvate dehydrogenase reaction is irreversible, acetyl-CoA cannot be converted to pyruvate, and hence animals cannot realize a net gain of carbohydrate from acetyl-CoA. Because plants have a glyoxylate cycle and animals do not, plants synthesize one molecule of succinate and one molecule of malate from two molecules of acetyl-CoA and one of oxaloacetate. The malate is converted to oxaloacetate, which reacts with another molecule of acetyl-CoA and thereby continues the reactions of the glyoxylate cycle. The succinate is also converted to oxaloacetate via the enzymes of the citric acid cycle. Thus, one molecule of oxaloacetate is diverted to carbohydrate synthesis and, therefore, plants are able to achieve net synthesis of carbohydrate from acetyl-CoA. 

Mercury interferes with mitochondrial oxidation in the brain through mercaptide formation with thiol groups in pyruvate oxidase. Succinic dehydrogenase of the citric acid cycle is also inhibited. 

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... 

Succinate is an intermediate of the citric acid cycle (and the glyoxylate cycle) produced by action of the enzyme succinyl-CoA synthetase on succinyl-CoA. Succinate is converted to fumarate by action of the enzyme succinate dehydrogenase (with formation of FADH2)... 

Succinyl-CoA is an intermediate of the citric acid cycle produced by decarboxylation of oi-ketoglutarate. The reaction is catalyzed by the a-ketoglutarate dehydrogenase enzyme complex. Succinyl-CoA is converted to succinate (with formation of GTP) in a reaction catalyzed by the enzyme succinyl-CoA synthetase. 

Succinate dehydrogenase, like aconitase, is an iron—sulfur protein. Indeed, succinate dehydrogenase contains three different kinds of iron—sulfur clusters, 2Fe-2S (two iron atoms bonded to two inorganic sulfides), 3Fe-4S, and 4Fe-4S. Succinate dehydrogenase— which consists of two subunits, one 70 kd and the other 27 kd—differs from other enzymes in the citric acid cycle in being embedded in the inner mitochondrial membrane. In fact, succinate dehydrogenase is directly associated with the electron-transport chain, the link between the citric acid cycle and ATP formation. FADH2 produced by the... 

Step 6. Formation of Fumarate—FAD-Linked Oxidation Succinate is oxidized to fumarate, a reaction that is catalyzed by the enzyme succinate dehydrogenase. This enzyme is an integral protein of the inner mitochondrial membrane. We shall have much more to say about the enzymes bound to the inner mitochondrial membrane in Ghapter 20. The other individual enzymes of the citric acid cycle are in the mitochondrial matrix. The electron acceptor, which is FAD rather than NADA is covalently bonded to the enzyme succinate dehydrogenase is also called a flavoprotein because of the presence of FAD with its flavin moiety. In the succinate dehydrogenase reaction, FAD is reduced to FADHo and succinate is oxidized to fumarate. 

Radioactive acetyl CoA can be generated by direct synthesis from C-acetate or from (3 oxidation of radioactive fatty acids, such as uniformly labeled palmitate. Examination of the reactions of the citric acid cycle reveals that neither of the two carbons that enter citrate horn acetate is removed as carbon dioxide during the first pass through the cycle. Labeled carbon from C-methyl-labeled acetate appears in C-2 and C-3 of oxaloacetate, because succinate is symmetrical, with either methylene carbon in that molecule labeling C-2 or C-3 of oxaloacetate. The conversion of oxaloacetate to phosphoenolpyruvate yields PEP labeled at C-2 or C-3 as well. Formation of glyceraldehyde 3-phosphate and its isomer dihydroxyacetone phosphate gives molecules, both labeled at carbons 2 and... 

Synthesis of succinyl-CoA in mammalian cells such as the red cell and liver cell can be accomplished either from a-KG or from succinate. The formation of succinyl-CoA occurs in the mitochondria as part of the citric acid cycle reactions. The requirement for a citric acid cycle to form ALA or protoporphyrin or heme has been shown by tracer studies with acetate and succinate [39], and by inhibition studies with malonate, Ira j -aconitate, fluoracetate, and arsenite [49]. The requirement for an electron transfer system from the citric acid cycle to O2 has been shown by inhibition studies with anaerobiosis and CO. The requirement for oxidative phosphorylation has been shown by dinitrophenol inhibition of ALA synthesis dinitrophenol may also inhibit ALA-synthetase [3,49]. 

In these anaerobic conditions, the citric acid cycle cannot be completed since the succinodehydro-genase activity requires the presence of FAD, a strictly respiratory coenzyme. The chain of reactions is therefore interrupted at succinate, which accumulates (0.5-1.5 g/1). The NADH generated by this portion of the Krebs cycle (from oxaloacetate to succinate) is reoxidized by the formation of glycerol from dihydroxyacetone. 

With the use of labeled succinate-1,4-C S succinate-2,3-C labeled a-ketoglutarate, and citrate as substrates for heme formation it was demonstrated that a-ketoglutarate gave rise to the succinyl derivative (27). By blocking succinic dehydrogenase with malonate (28) the succinyl derivative was found to be formed via the citric acid cycle, or to a lesser extent more directly from succinate. The predicted carbon atoms in heme contained the C . 

This normally requires the functioning ot a series of enzyme systems within the cell. The formation of succinyl CoA requires a citric acid cycle as shown by Shemin s tracer studies with succinate-1,4-C and succinate-2,3-C this was also shown by inhibition studies with malonate, trans-aconitate, fluoroacetate, or arsenite (35). Coupled to the citric acid cycle is an electron transfer system to O as shown by inhibition by CO and anaerobiosis. Inhibition by dinitrophenol su ests that oxidative phosphorylation is required, although dinitrophenol may inhibit 5-AL synthetase more directly (36). For AL synthesis, pyridoxal-P is required as shown by Schulman and Richert (37) on vitamin B -dehcient chicks and by inhibitors of pyridoxal-P, e.g., deoxypyridoxine, isonicotinic hydrazide, etc. (35). 

In erythrocytes the main pathway for succinyl CoA formation appears to be from a-ketoglutaric acid by oxidation via the citric acid cycle, through TPP and lipoic acid, to form succinyl lipoate which with CoA forms succinyl CoA. The conversion of succinate to succinyl CoA has been shown by Shemin and Kumin (38) who used labeled succinate in the presence of malonate. 

The most significant aspect of the citric-acid cycle is the process by which ATP formation from ADP and inorganic phosphate is tied up with electron transfer reactions. The oxidation of a-ketoglutarate to succinate by oxygen involves the esterification of four molecules of inorganic phosphate per atom of oxygen. - It has been assumed that four oxidoreduc-... 

The efficiency of an enzyme can be reduced or can even become negligible in the presence of certain substances, known as inhibitors. Many inhibitors have structural resemblances with the substrates and compete with them for the formation of complexes with the enzyme. This is the case of the inactivation of cytochrome c oxidase by the cyanide ion, which blocks the mitochondrial electron-transport chain to oxygen. Similarly, the inactivation of the succinate dehydrogenase by malonate involves its inhibition of the conversion of succinate to fumarate in the citric acid cycle. In the latter case, the mechanism for competitive inhibition is... 

From the previous section it is evident that our knowledge about the respiratory chain is still quite incomplete. We know which prosthetic groups participate (cf. diagram in Section 4). It remains to be clarified, however, to what proteins they are bound and what role the metals and any new cofactors might play. The reason for this unsatisfactory state of knowledge is that the enzymes under consideration are bound very firmly to the mitochondrial structure (cf. Chapt. XIX-3). Only very recently have techniques been developed to subdivide the mitochondria in such a manner that most of their activity is retained. The subunits thus obtained have been called electron-transport particles (Green and co-workers). Some of the catalytic capabilities have been sacrificed (e.g. the enzymes of the citric acid cycle). But they are still able to oxidize NADHs or succinate with consumption of Oj and formation of ATP (see below). With the further destruction of these subunits, the capacity for oxidative phosphorylation disappears. 

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