Citrate aconitase reaction

FIGURE 20.7 (a) The aconitase reaction converts citrate to cis-aconitate and then to isocitrate. Aconitase is stereospecific and removes the pro-/ hydrogen from the pro-/ arm of citrate, (b) The active site of aconitase. The iron-sulfur cluster (red) is coordinated by cysteines (yellow) and isocitrate (white). 

Why did nature use an Fe-S cluster to catalyze this reaction, when an enzyme such as fumarase can catalyze the same type of chemistry in the absence of any metals or other cofactors One speculation would be that since aconitase must catalyze both hydrations and dehydrations, and bind substrate in two orientations, Fe in the comer of a cubane cluster may provide the proper coordination geometry and electronics to do all of these reactions. Another possibility is that the cluster interconversion is utilized in vivo to regulate enzyme activity, and thus, help control cellular levels of citrate. A third, but less likely, explanation is that during evolution an ancestral Fe-S protein, whose primary function was electron transfer, gained the ability to catalyze the aconitase reaction through random mutation. 

Potent metabolic inhibitors of the citric acid cycle. Fluo-roacetate (F-CH2COO ) must first be converted to flu-oroacetyl-S-CoA (by acetyl-CoA synthetase) and thence to fluorocitrate (by citrate synthase) before it can act as a potent metabohc inhibitor of the aconitase reaction as well as citrate transport. Submicromolar concentrations of ( )-erythro-Q iOTOcitTate can irreversibly inhibit citrate uptake by isolated brain mitochondria. 

There are two Krebs cycle inhibitors that are worth mentioning. Malonate inhibits succinate dehydrogenase because of its very similar structure. Fluoro-acetate inhibits cis-aconitase, which is an Fe-S enzyme. The fluoroacetate replaces acetate as a substrate in the citrate synthase reaction when this combines with cis-aconitase, however, no further reaction becomes possible. 

See also Aconitase, Fluoroacetate, Citrate Synthase, Reaction Diagram, Acetate Thiokinase... 

It exerts its effect by being an inhibitor of the Krebs cycle. Fluoroacetate substitutes for acetate and is converted to fluoroacetyl CoA. This is condensed with oxaloacetate to produce fluorocitrate in other words citrate synthase uses fluoroacetate as a substrate and forms fluorocitrate that inhibits aconitase (reaction 2 in Fig. 10-9A and B). If the Krebs cycle is not functioning correctly, the rate of NADH production will be insufficient to maintain the mitochondrial proton gradient, and the ATP concentration will decline. A further complication is that processes such as the Krebs cycle have an important role in regenerating moiety carrier molecules. If the cycle is inhibited, then vital carriers such as oxaloacetate and coenzyme A become depleted. 

Citrate is isomerized to isocitrate by aconitase in a two-step process involving aconitate as an intermediate (Figure 20.7). In this reaction, the elements... 

Aconitase catalyzes the citric acid cycle reaction citrate isocitrate... 

Step 2 of Figure 29.12 Isomerization Citrate, a prochiral tertiary alcohol, is next converted into its isomer, (2, 35)-isocitrate, a chiral secondary alcohol. The isomerization occurs in two steps, both of which are catalyzed by the same aconitase enzyme. The initial step is an ElcB dehydration of a /3-hydroxy acid to give cfs-aconitate, the same sort of reaction that occurs in step 9 of glycolysis (Figure 29.7). The second step is a conjugate nucleophilic addition of water to the C=C bond (Section 19.13). The dehydration of citrate takes place specifically on the pro-R arm—the one derived from oxaloacetate—rather than on the pro-S arm derived from acetyl CoA. 

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase) the reaction occurs in two steps dehydration to r-aconitate, some of which remains bound to the enzyme and rehydration to isocitrate. Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is due to channeling— transfer of the product of citrate synthase directly onto the active site of aconitase without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. The poison fluo-roacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate. 

Aconitase, an unstable enzyme,4 is concerned with the reversible conversion of cis-aconitate to either citric acid or isocitric acid. It may be noted that the entire system of tricarboxylic cycle enzymes are present in the mitochondria separated from cells, and, furthermore, it has been found that the mitochondrial enzymes differ from the isolated enzymes in that the former require no addition of D.P.N. (co-enzyme I) or T.P.N. (co-enzyme II) for activity. Peters suggests that the citrate accumulation is caused by the competitive reaction of the fluorocitrate with aconitase required for the conversion of citrate to isocitrate. This interference with the tricarboxylic acid... 

Fig. 9. Pathway duplication the methyl citrate cycle and the glyoxylate shunt. A pathway for acetate metabolism in E. coli that uses the glyoxylate shunt is depicted on the right. Part of the methyl citrate cycle, a pathway for propionate metabolism, is depicted on the left. The pathways are analogous furthermore, three of the four steps are catalyzed by homologous enzymes. PrpE (propionyl-CoA synthase) is homologous to AcsA (acetyl-CoA synthase). PrpC (2-methyl-citrate synthase) is homologous to GltA (citrate synthase). PrpB (2-methyl-isocitrate lyase) is homologous to AceA (isocitrate lyase). The third step in the methyl citrate cycle has been suggested to be catalyzed by PrpD the second half of the reaction (the hydration) can be catalyzed by aconitase.

Aconitase [citrate(isocitrate) hydro-lyase, EC 4.2.1.3] is the second enzyme of the citric acid cycle, which plays a central role in metabolism for all aerobic organisms. This enzyme catalyzes a dehydration-rehydration reaction interconverting citrate and 2R,3S-isocitrate via the allylic intermediate ds-aconitate. 

Aconitase was first described 50 years ago by Martius (1,2) and soon there after named by Breusch (3). The enzyme demonstrated the then surprizing ability to distinguish between the chemicadly identical acetyl arms of citrate (4). The stereo-specificity of enzyme catalyzed reactions was not fully understood until the late 1940 s when Ogston point out that as long as a substrate attaches to an asymmetric enzyme at three points, the enzyme can differentiate between two identical amis of a symmetrical molecule (5). 

Citrate rearranges to isocitrate in a reaction catalyzed by aconitase. 

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. 

Two consecutive reactions of the citric acid cycle (Fig. 10-6), the dehydration of citrate to form czs-aconi-tate and the rehydration in a different way to form isocitrate (Eq. 13-17), are catalyzed by aconitase (aconi-tate hydratase). Both reactions are completely stereospecific. In the first (Eq. 13-17, step a), the pro-R proton from C-4 (stereochemical numbering) of citrate is removed and in step c isocitrate is formed. Proton addition is to the re face in both cases. 

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. 

Aconitase is the trivial name for citrate dehydratase cw-aconitate hydratase (EC 4.2.1.3). It catalyzes the reversible isomerization reaction of citrate into isocitrate via the intermediate cA-aconitate (Figure 2). It is a water-soluble, monomeric protein. In eukaryotic cells aconitase is located in the mitochondrial matrix. In prokaryotes the enzyme occurs in the cytoplasma. The pig heart enzyme consists of 754 amino-acid residues, providing a molecular mass of 83 kDa [27], Aconitase from other sources has similar size. The porcine protein is synthesized with a mitochondrial targeting sequence. The mature, functional protein can be (over)expressed in Escherichia coli [28],... 

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 ... 

Step 2 involves the dehydration of citrate to cir-aconitate followed by the hydration of cis-aconitate to isocitrate. Aconitase catalyzes these reversible reactions. 

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