Oxaloacetate removal from citric acid cycle

Figure 10-6 Reactions of the citric acid cycle (Krebs tricarboxylic acid cycle). Asterisks designate positions of isotopic label from entrance of carboxyl-labeled acetate into the cycle. Note that it is not the two carbon atoms from acetyl-CoA that are immediately removed as C02 but two atoms from oxaloacetate. Only after several turns of the cycle are the carbon atoms of the acetyl-CoA completely converted to C02. Nevertheless, the cycle can properly be regarded as a mechanism of oxidation of acetyl groups to C02. Green daggers (+) designate the position of 2H introduced into malate as 2H from the medium. FADS designates covalently bound 8-histidyl-FAD (see Chapter 15).

One of the simplest biochemical addition reactions is the hydration of carbon dioxide to form carbonic acid, which is released from the zinc-containing carbonic anhydrase (left, Fig. 13-1) as HC03-. Aconitase (center, Fig. 13-4) is shown here removing a water molecule from isocitrate, an intermediate compound in the citric acid cycle. The H20 that is removed will become bonded to an iron atom of the Fe4S4 cluster at the active site as indicated by the black H20. An enolate anion derived from acetyl-CoA adds to the carbonyl group of oxaloacetate to form citrate in the active site of citrate synthase (right, Fig. 13-9) to initiate the citric acid cycle. 

It may be protested that the reaction of the citric acid cycle by which oxaloacetate is converted to oxo-glutarate does not follow exactly the pattern of Fig. 17-18. The carbon dioxide removed in the decarboxylation step does not come from the part of the molecule donated by the acetyl group but from that formed from oxaloacetate. However, the end result is the same. Furthermore, there are two known citrate-forming enzymes with different stereospecificities (Chapter 13), one of which leads to a biosynthetic pathway strictly according to the sequence of Fig. 17-18. 

The cycle oxidizes acetyl-CoA, and to perform this task, it must convert acetyl-CoA to citrate. For this to be achieved, oxaloacetate must be available. If the removal of intermediates results in a decrease in the amount of oxaloacetate for this purpose, acetyl-CoA cannot be removed and will accumulate. This will inhibit the pyruvate dehydrogenase complex and activate pyruvate carboxylase, leading to the conversion of pyruvate to oxaloacetate. This product is now available to condense with the acetyl-CoA to produce citrate, which will restore the status quo. Reactions like that of pyruvate carboxylase that provide molecules for the replacement of intermediates of the citric acid cycle are known as anaplerotic reactions (Greek, meaning to fill up ana = up + plerotikos from pleroun = to make full ). 

We now turn to the fates of the carbon skeletons of amino acids after the removal of the a-amino group. The strategy of amino acid degradation is to transform the carbon skeletons into major metabolic intermediates that can be converted into glucose or oxidized by the citric acid cycle. The conversion pathways range from extremely simple to quite complex. The carbon skeletons of the diverse set of 20 fundamental amino acids are furmeled into only seven molecules pyruvate, acetyl CoA, acetoacetyl CoA, a-ketoglutarate, succinyl CoA, fumarate, and oxaloacetate. We see here a striking example of the remarkable economy of metabolic conversions, as well as an illustration of the importance of certain metabolites. 

A FIGURE 8-9 The citric acid cycle, in which acetyl groups transferred from acetyl CoA are oxidized to CO2. In reaction 1, a two-carbon acetyl residue from acetyl CoA condenses with the four-carbon molecule oxaloacetate to form the six-carbon molecule citrate. In the remaining reactions (2-9) each molecule of citrate is eventually converted back to oxaloacetate, losing two CO2 molecules in the process. In each turn of the cycle, four pairs of electrons are removed from carbon atoms, forming... 

Finally, oxaloacetate is simultaneously decarboxylated and phosphorylated by phosphoenolpyruvate carboxykinase in the cytosol. The CO2 that was added to pyruvate by pyruvate carboxylase comes off in this step. Recall that, in glycolysis, the presence of a phosphoryl group traps the unstable enol isomer of pyruvate as phosphoenolpyruvate (Section 16.1.7). In gluconeogenesis, the formation of the unstable enol is driven by decarboxylation—the oxidation of the carboxylic acid to CO2—and trapped by the addition of a phosphate to carbon 2 from GTP. The two-step pathway for the formation of phosphoenolpyruvate from pyruvate has a AG° of + 0.2 kcal mol ( + 0.13 kj moP ) in contrast with +7.5 kcal mol ( + 31 kj mol ) for the reaction catalyzed by pyruvate kinase. The much more favorable AG° for the two-step pathway results from the use of a molecule of ATP to add a molecule of CO2 in the carboxylation step that can be removed to power the formation of phosphoenolpyruvate in the decarboxylation step. Decarboxylations often drive reactions otherwise highly endergonic. This metabolic motif is used in the citric acid cycle (Section IS.x.x), the pentose phosphate pathway (Section 17.x.x), and fatty acid synthesis (Section 22.x.x). 

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... 

In the first reaction of the citric acid cycle, acetyl-CoA reacts with oxaloacetate to form citrate. The mechanism for the reaction shows that an aspartate side chain of the enzyme removes a proton from the a-carbon of acetyl-CoA, creating an enolate ion. This enolate ion adds to the keto carbonyl carbon of oxaloacetate and the carbonyl oxygen picks up a proton from a histidine side chain. This is similar to an aldol addition where the a-carbanion (enolate ion) of one molecule is the nucleophile and the carbonyl carbon of another is the electrophile (Section 18.10). The intermediate (a thioester) that results is hydrolyzed to citrate in a nucleophilic addition-elimination reaction (Section 16.9). 

Pyruvate carboxylase catalyses the conversion of pyruvate to oxaloacetate using ATP and CO2. This is an important reaction both for gluconeogenesis (to bypass the pyruvate kinase reaction) and also for the normal function of the citric acid cyde. For the citric acid cycle to begin, one molecule each of oxaloacetate and acetyl CoA is required. If there is a shortage of oxaloacetate, the balance is restored by the action of pyruvate carboxylase. If, for example, oxaloacetate has been removed from the cycle to enter the amino acid synthesis pathways, it can simply be regenerated fiom pyruvate (31b). 

There are a total of eight reactions and eight enzymes in the citric acid cycle. Initially, an acetyl gronp (2C) from acetyl-CoA bonds with oxaloacetate (4C) to yield citrate (6C) (see Figure 18.11). Then, two decarboxylation reactions remove carbon atoms as CO2 molecules to give succinyl-CoA (4C). Finally, a series of reactions converts four-carbon succinyl-CoA to oxaloacetate, which combines with another acetyl-CoA, and the citric cycle starts aU over, hi one turn of the citric acid cycle, four oxidation reactions provide hydrogen ions and electrons, which are used to reduce FAD and NAD+ coenzymes (see Figure 18.12). 

FIGURE 18.11 In the citric acid cycle, two carbon atoms are removed as CO2 from six-carbon citrate to give four-carbon succinyl-CoA, which is converted to four-carbon oxaloacetate. 

We have seen that the citric acid cycle begins when a two-carbon acetyl group from acetyl-CoA combines with oxaloacetate to form citrate. Through oxidation and reduction, two carbon atoms are removed from citrate to yield two CO2 and a four-carbon compound that undergoes reactions to regenerate oxaloacetate. 

---