Steps in the TCA Cycle

The TCA cycle. In each turn of the cycle, acetyl-CoA from the glycolytic pathway or from /3 oxidation of fatty acids enters and two fully oxidized carbon atoms leave (as C02). ATP is generated at one point in the cycle, and coenzyme molecules are reduced. The two C02 molecules lost in each cycle originate from the oxaloacetate of the previous cycle rather than from incoming acetyl from acetyl-CoA. This point is emphasized by the use of color. 

---

The next step in the TCA cycle, the oxidation of succinate to fumarate, involves insertion of a double bond into a saturated hydrocarbon chain. 

Starting with citrate, isocitrate, u-ketoglntarate, and succinate, state which of the individual carbons of the molecule undergo oxidation in the next step of the TCA cycle. Which molecules undergo a net oxidation ... 

In essence, this series of four reactions has yielded a fatty acid (as a CoA ester) that has been shortened by two carbons, and one molecule of acetyl-CoA. The shortened fatty acyl-CoA can now go through another /3-oxidation cycle, as shown in Figure 24.10. Repetition of this cycle with a fatty acid with an even number of carbons eventually yields two molecules of acetyl-CoA in the final step. As noted in the first reaction in Table 24.2, complete /3-oxidation of palmitic acid yields eight molecules of acetyl-CoA as well as seven molecules of FADHg and seven molecules of NADFI. The acetyl-CoA can be further metabolized in the TCA cycle (as we have already seen). Alternatively, acetyl-CoA can also be used as a substrate in amino acid biosynthesis (Chapter 26). As noted in Chapter 23, however, acetyl-CoA cannot be used as a substrate for gluco-neogenesis. 

Ketone body synthesis occurs only in the mitochondrial matrix. The reactions responsible for the formation of ketone bodies are shown in Figure 24.28. The first reaction—the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA—is catalyzed by thiolase, which is also known as acetoacetyl-CoA thiolase or acetyl-CoA acetyltransferase. This is the same enzyme that carries out the thiolase reaction in /3-oxidation, but here it runs in reverse. The second reaction adds another molecule of acetyl-CoA to give (i-hydroxy-(i-methyl-glutaryl-CoA, commonly abbreviated HMG-CoA. These two mitochondrial matrix reactions are analogous to the first two steps in cholesterol biosynthesis, a cytosolic process, as we shall see in Chapter 25. HMG-CoA is converted to acetoacetate and acetyl-CoA by the action of HMG-CoA lyase in a mixed aldol-Claisen ester cleavage reaction. This reaction is mechanistically similar to the reverse of the citrate synthase reaction in the TCA cycle. A membrane-bound enzyme, /3-hydroxybutyrate dehydrogenase, then can reduce acetoacetate to /3-hydroxybutyrate. 

The situation is simpler for odd numbered fatty acyl derivatives as [3-oxidation proceeds normally until a 5-carbon unit remains, rather than the usual 4-carbon unit. The C5 moiety is cleaved to yield acetyl-CoA (C2) and propionyl-CoA (C3). Propionyl CoA can be converted to succinyl CoA and enter the TCA cycle so the entire molecule is utilized but with a slight reduction in ATP yield as the opportunity to generate two molecules of NADH by isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase is lost because succinyl-CoA occurs after these steps in the Krebs cycle (Figure 7.18). 

The first step of the TCA cycle is the reaction catalyzed by citrate synthase, in which acetyl-CoA enters the cycle and citrate is formed. 

The glyoxylate cycle permits growth on a two-carbon source. The glyoxylate cycle bypasses the two steps of the TCA cycle in which C02 is released. Furthermore, two molecules of acetyl-CoA are taken in per turn of the cycle rather than just one, as in the TCA cycle. The net result is the conversion of two mole-... 

This step leaves two cleavage products. The first, derived from the two carbons at the carboxyl end of the fatty acid, is acetyl-CoA, which can be further metabolized in the TCA cycle. The second cleavage product is a shorter fatty acyl-CoA. Thus, for example, the initial step of digesting a fatty acid with 16 carbons is an acyl-CoA molecule where the acyl group has 14 carbons and a molecule of acetyl-CoA. The P-oxidation scheme may be used to accommodate unsaturated fatty acids also. The reactions occur as described previously for the saturated portions of the molecule. Where a trans carbon-carbon double bond occurs between the %- and p-carbons of the acyl-CoA, the accommodation is fairly simple reaction 1 isn t needed. Where the double bonds are in the cis configuration, or are between the P and y carbons, isomerase enzymes change the location of the double bonds to make recognizable substrates for P-oxidation. 

Fatty acid biosynthesis (and most biosynthetic reactions) requires NADPH to supply the reducing equivalents. Oxaloacetate is used to generate NADPH for biosynthesis in a two-step sequence. The first step is the malate dehydrogenase reaction found in the TCA cycle. This reaction results in the formation of NAD from NADH (the NADH primarily comes from glycolysis). The malate formed is a substrate for the malic enzyme reaction, which makes pyruvate, CO2, and NADPH. Pyruvate is transported into the mitochondria where pyruvate carboxylase uses ATP energy to regenerate oxaloacetate. 

Figure 20-4. Biochemical pathways for gluconeogenesis in the liver. Alanine, a major gluconeogenic substrate, is used to synthesize oxaloacetate. The carbon skeletons of glutamine and other glucogenic amino acids feed into the TCA cycle as a-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate and thus also provide oxaloacetate. Conversion of oxaloacetate to phosphoenolpyruvate and ultimately to glucose limits the availability of oxaloacetate for citrate synthesis and thus greatly diminishes flux through the initial steps of the TCA cycle (dashed lines). Concurrent P-oxidation of fatty acids provides reducing equivalents (NADH and FADH2) for oxidative phosphorylation but results in accumulation of acetyl-CoA.

In the next step of the TCA cycle, the hydroxyl (alcohol) group of citrate is moved to an adjacent carbon so that it can be oxidized to form a keto group. The isomerization of citrate to isocitrate is catalyzed by the enzyme aconitase, which is named for an intermediate of the reaction. The enzyme isocitrate dehydrogenase catalyzes the oxidation of the alcohol group and the subsequent cleavage of the carboxyl group to release CO2 (an oxidative decarboxylation). 

Up to this stage of the TCA cycle, two carbons have been stripped of their available electrons and released as CO2. Two pairs of these electrons have been transferred to 2 NAD, and one GTP has been generated. However, two additional pairs of electrons arising from acetyl CoA still remain in the TCA cycle as part of succinate. The remaining steps of the TCA cycle transfer these two pairs of electrons to FAD and NAD and add H2O, thereby regenerating oxaloacetate. 

Isocitrate dehydrogenase releases the first CO2, and a-ketoglutarate dehydrogenase releases the second CO2. There is no net consumption of oxaloacetate in the TCA cycle— the first step use an oxaloacetate, and the last step produces one. The utilization and regeneration of oxaloacetate is the "cycle" part of the TCA cycle. 

Acetyl CoA serves as a common point of convergence for the major pathways of fuel oxidation. It is generated directly from the (3-oxidation of fatty acids and degradation of the ketone bodies (3-hydroxybutyrate and acetoacetate (Fig. 20.14). It is also formed from acetate, which can arise from the diet or from ethanol oxidation. Glucose and other carbohydrates enter glycolysis, a pathway common to all cells, and are oxidized to pyruvate. The amino acids alanine and serine are also converted to pyruvate. Pyruvate is oxidized to acetyl CoA by the pyruvate dehydrogenase complex. A number of amino acids, such as leucine and isoleucine are also oxidized to acetyl CoA. Thus, the final oxidation of acetyl CoA to CO2 in the TCA cycle is the last step in all the major pathways of fuel oxidation. 

The TD-induced inhibition of metabolic flux through PDHC takes place in all cells of the affected organism (Jankowska-Kulawy et al. 2010). It causes primary inhibition of acetyl-CoA synthesis in the mitochondria, yielding a proportional reduction of its metabolic flux through first steps of the TCA cycle and further aggravation of this suppression due to inhibition of KDHC (Jankowska-Kulawy et al. 2010 Shi et al. 2007) (Figure 33.1). As a result, oxidative phosphorylation process slows down, yielding ATP deficits (Jhala and Hazell 2011). 

Pyruvate produced by glycolysis is a significant source of acetyl-CoA for the TCA cycle. Because, in eukaryotic ceils, glycolysis occurs in the cytoplasm, whereas the TCA cycle reactions and ail subsequent steps of aerobic metabolism take place in the mitochondria, pyruvate must first enter the mitochondria to enter the TCA cycle. The oxidative decarboxylation of pyruvate to acetyl-CoA,... 

Citrate synthase is the first step in this metabolic pathway, and as stated the reaction has a large negative AG°. As might be expected, it is a highly regulated enzyme. NADH, a product of the TCA cycle, is an allosteric inhibitor of citrate synthase, as is succinyl-CoA, the product of the fifth step in the cycle (and an acetyl-CoA analog). 

The TCA cycle can now be completed by converting succinate to oxaloacetate. This latter process represents a net oxidation. The TCA cycle breaks it down into (consecutively) an oxidation step, a hydration reaction, and a second oxidation step. The oxidation steps are accompanied by the reduction of an [FAD] and an NAD. The reduced coenzymes, [FADHg] and NADH, subsequently provide reducing power in the electron transport chain. (We see in Chapter 24 that virtually the same chemical strategy is used in /3-oxidation of fatty acids.)... 

There are several examples in which metabolites that toxify the organism responsible for their synthesis are produced. The classic example is fluoroacetate (Peters 1952), which enters the TCA cycle and is thereby converted into fluorocitrate. This effectively inhibits aconitase—the enzyme involved in the next metabolic step—so that cell metabolism itself is inhibited with the resulting death of the cell. Walsh (1982) has extensively reinvestigated the problan and revealed both the complexity of the mechanism of inhibition and the stereospecihcity of the formation of fluorocitrate from fluoroacetate (p. 239). It should be noted, however, that bacteria able to degrade fluoroacetate to fluoride exist so that some organisms have developed the capability for overcoming this toxicity (Meyer et al. 1990). 

A carrier molecule containing four carbon atoms (the C4 unit) takes up a C2 unit (the activated acetic acid ), which is introduced into the cycle. The product is a six-carbon molecule (the C6 unit), citric acid, or its salt, citrate. CO2 is cleaved off in a cyclic process, so that a C5 unit is left this loses a further molecule of CO2 to give the C4 unit, oxalacetate. In the living cell, this process involves ten steps, which are catalysed by eight enzymes. However, the purpose of the TCA cycle is not the elimination of CO2, but the provision of reduction equivalents, i.e., of electrons, and... 

---