Citrate in the citric acid cycle

While CoA was discovered as the "acetylation coenzyme," it has a far more general function. It is required, in the form of acetyl-CoA, to catalyze the synthesis of citrate in the citric acid cycle. It is essential to the P oxidation of fatty acids and carries propionyl and other acyl groups in a great variety of other metabolic reactions. About 4% of all known enzymes require CoA or one of its esters as a substrate.4... 

Cis-aconitate is an intermediate in synthesis of iso citrate from citrate in the citric acid cycle. The enzyme catalyzing the reaction is aconitase. 

You might note that C2 of glycerol is a prochiral center with two identical arms, a situation similar to that of citrate in the citric acid cycle (Section 22.4). As is typical for enzyme-catalyzed reactions, the phosphorylation of glycerol is selective. Only the pro-R arm undergoes reaction, although this can t be predicted in advance. 

The reaction that catalyses the synthesis of citrate in the citric acid cycle is ... 

Glycolysis and the citric acid cycle (to be discussed in Chapter 20) are coupled via phosphofructokinase, because citrate, an intermediate in the citric acid cycle, is an allosteric inhibitor of phosphofructokinase. When the citric acid cycle reaches saturation, glycolysis (which feeds the citric acid cycle under aerobic conditions) slows down. The citric acid cycle directs electrons into the electron transport chain (for the purpose of ATP synthesis in oxidative phosphorylation) and also provides precursor molecules for biosynthetic pathways. Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated. 

Elucidating the stereochemistry of reaction at prochirality centers is a powerful method for studying detailed mechanisms in biochemical reactions. As just one example, the conversion of citrate to (ds)-aconitate in the citric acid cycle has been shown to occur with loss of a pro-R hydrogen, implying that the reaction takes place by an anti elimination mechanism. That is, the OH and H groups leave from opposite sides of the molecule. 

The first step in the citric acid cycle is reaction of oxaloacetate with acetyl CoA to give citrate. Propose a mechanism, using acid or base catalysis as needed. 

Enzymes work by bringing reactant molecules together, holding them, in the orientation necessary for reaction, and providing any necessary acidic or basic sites to catalyze specific steps. As an example, let s look at citrate synthase, an enzyme that catalyzes the aldol-like addition of acetyl CoA to oxaloacetate to give citrate. The reaction is the first step in the citric acid cycle, in which acetyl groups produced by degradation of food molecules are metabolized to yield C02 and H20. We ll look at the details of the citric acid cycle in Section 29.7. 

This diagram shows the citric acid cycle. For simplicity, the molecules in the citric acid cycle are represented here as acids. In the body, they are actually present as iens. For example, citric acid is present as citrate ions. 

One of the first persons to study the oxidation of organic compounds by animal tissues was T. Thunberg, who between 1911 and 1920 discovered about 40 organic compounds that could be oxidized by animal tissues. Salts of succinate, fumarate, malate, and citrate were oxidized the fastest. Well aware of Knoop s (3 oxidation theory, Thunberg proposed a cyclic mechanism for oxidation of acetate. Two molecules of this two-carbon compound were supposed to condense (with reduction) to succinate, which was then oxidized as in the citric acid cycle to oxaloacetate. The latter was decarboxylated to pyruvate, which was oxidatively decarboxylated to acetate to complete the cycle. One of the reactions essential for this cycle could not be verified experimentally. It is left to the reader to recognize which one. 

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. 

Among the most deadly of simple compounds is sodium fluoroacetate. The LD50 (the dose lethal for 50% of animals receiving it) is only 0.2 mg/kg for rats, over tenfold less than that of the nerve poison diisopropylphosphofluoridate (Chapter 12).a b Popular, but controversial, as the rodent poison "1080," fluoroacetate is also found in the leaves of several poisonous plants in Africa, Australia, and South America. Surprisingly, difluoroacetate HCF2-COO is nontoxic and biochemical studies reveal that monofluoroacetate has no toxic effect on cells until it is converted metabolically in a "lethal synthesis" to 2R,3R-2-fluorocitrate, which is a competitive inhibitor of aconitase (aconitate hydratase, Eq. 13-17).b This fact was difficult to understand since citrate formed by the reaction of fluorooxalo-acetate and acetyl-CoA has only weak inhibitory activity toward the same enzyme. Yet, it is the fluorocitrate formed from fluorooxaloacetate that contains a fluorine atom at a site that is attacked by aconitase in the citric acid cycle. 

Answer In the citric acid cycle, the entering acetyl-CoA combines with oxaloacetate to form citrate. One turn of the cycle regenerates oxaloacetate and produces two C02 molecules. There is no net synthesis of oxaloacetate in the cycle. If any cycle intermediates are channeled into biosynthetic reactions, replenishment of oxaloacetate is essential. Four enzymes can... 

Answer Anaplerotic reactions replenish intermediates in the citric acid cycle. Net synthesis of a-ketoglutarate from pyruvate occurs by the sequential actions of (1) pyruvate carboxylase (which makes extra molecules of oxaloacetate), (2) pyruvate dehydrogenase, and the citric acid cycle enzymes (3) citrate synthase, (4) aconitase, and (5) isocitrate dehydrogenase ... 

Another example occurs in the citric acid cycle, where the enzyme aconitase catalyzes the elimination of water from citrate to produce aconitate ... 

The overall consumption of one molecule of acetyl-CoA in the citric acid cycle is an exergonic process AG° = —60 kJ mol-1. All but two of the individual reactions are exergonic. Step 2 (citrate— isocitrate) and step 8 (malate —>oxaloacetate) are endergonic (Fig. 12-3). 

There are four major regulatory enzymes in the citric acid cycle. These are citrate synthase (step 1), isocitrate dehydrogenase (step 3), 2-oxoglutarate dehydrogenase (step 4), and succinate dehydrogenase (step 6). 

The dehydration of citrate to yield n s-aconitatc, a step in the citric acid cycle, involves thepio-R "arm" of citrate rather than the pro-S arm. Which of the following two products is formed ... 

Perhaps the best characterized example of a subsite differentiated [4Fe-4S] protein is aconitase, which catalyzes the citrate-isocitrate isomerization in the citric acid cycle (257). Aconitase isolated aerobically is inactive and contains a [3Fe-4S] cluster. Activity is restored by incubation with Fe and this also reconstitutes the [4Fe-4S] cluster. Oxidation of the core results in loss of the fourth iron atom, regenerating the [3Fe-4S] form. Mossbauer studies have demonstrated that only one of the four iron sites is exchanged (258). X-ray studies on both [3Fe-4S] and [4Fe-4S] forms of pig heart aconitase 258a) showed that insertion of iron into [3Fe-4S] occurs isomorphously. The positions of the common atoms in the two forms of the core agree to within 0.1 A, supporting the view of the [3Fe-4S] cluster as an iron-voided cubane. A similar result was obtained for the seven iron ferredoxin from Azo-... 

Note that the standard free energy for this reaction, unlike that for the other steps in the citric acid cycle, is significantly positive. The oxidation of malate is driven by the utilization of the products—oxaloacetate by citrate synthase and NADH by the electron-transport chain. 

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