Citrate to Isocitrate

In this reaction, the tertiary alcoholic group of citrate is converted to a secondary alcoholic group that is more readily oxidized. The isomerization catalyzed by aconi-tate dehydratase (or aconitase) occurs by removal and addition of water with the formation of an intermediate, c -aconitate  

The cA-aconitate may not be an obligatory intermediate, since the enzyme presumably can isomerize 

Although citrate is a symmetrical molecule, its two -CH2COOH groups are not identical with regard to the orientation of the -OH and -COOH groups. Thus, citrate reacts in an asymmetrical manner with the enzyme, so that only the -CH2COOH group derived from oxaloacetate is modified. A three-point attachment of the enzyme to the substrate accounts for the asymmetrical nature of the reaction. 

A powerful competitive inhibitor (highly toxic) of aconitate dehydratase is fluorocitrate, an analogue of citrate  

In vivo, fluorocitrate can be formed by condensation of fluoroacetyl-CoA with oxaloacetate by action of citrate synthase. Fluoroacetyl-CoA itself is synthesized from flu-oroacetate by acetyl-CoA synthase. 

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Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

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... 

The catalytic efficiency of this enzyme to hydrolyze 5-fluoro-5,6-dihydro-uracil was found to be approximately twice that toward 5,6-dihydrouracil [152], 2-Fluoro-/3-alanine can either be eliminated via the bile after conjugation with bile acids, or be converted to fluoroacetate (4.238) [153], The latter metabolite is transformed to fluorocitrate, a potent inhibitor of the aconi-tase-catalyzed conversion of citrate to isocitrate. This inhibition probably explains the clinical neurotoxicity of 5-fluorouracil [154] [155],... 

Fluoroacetate produces its toxic action by inhibiting the citric acid cycle. The fluorine-substituted acetate is metabolized to fluoroci-trate that inhibits the conversion of citrate to isocitrate. There is an accumulation of large quantities of citrate in the tissue, and the cycle is blocked. The heart and central nervous system are the most critical tissues involved in poisoning by a general inhibition of oxidative energy metabolism. ... 

Citrate Synthase Is the Gateway to the TCA Cycle Aconitase Catalyzes the Isomerization of Citrate to Isocitrate... 

The equilibrium constant for the conversion of citrate to isocitrate is small, and the interconversion is rapid, so these... 

Bergner-Lang (104) reported that 74% of 80 commercial orange juice samples tested failed to meet the citrate-to-isocitrate ratio of a series of laboratory-prepared orange juices, and 87% of the same samples were below the minimum for fresh juice. 

These conclusions were challenged by Rother (108) as inappropriate due to the high percentage of Brazilian orange juice in the commercial products. He presented data where 17 of 19 samples fell within the normal citrate-to-isocitrate ratio range of 50 to 170. 

Both of these reactions are followed by exergonic reactions. The equilibrium of the reaction malate oxaloacetate (step 8) lies in favor of malate formation, so at equilibrium the concentration of oxaloacetate will be low. The next reaction in the cycle (oxaloacetate + acetyl-CoA — citrate) (step 1) is, however, exergonic, and the oxaloacetate is removed to condense with acetyl-CoA. Similarly, the conversion of citrate to isocitrate is endergonic, and at equilibrium the reaction favors the formation of citrate. The next reaction in the cycle (isocitrate—>2-oxoglutarate) is exergonic, and so the isocitrate is removed thus allowing this reaction to proceed. 

Fluoroacetate is converted to fluoroacetyl-CoA. This substance then reacts with oxaloacetate to produce fluorocitrate. Fluorocitrate is toxic because it inhibits aconitase, the enzyme that normally converts citrate to isocitrate, hence the buildup of citrate. In the plant fluoroacetate is stored, in vacuoles away from the mitochondria. 

In some cases, separate proteins have been fused together at the genetic level to create a single multidomain, multifunctional enzyme (Figure 3-20c). For instance, the isomerization of citrate to isocitrate in the citric acid cycle is catalyzed by aconitase, a single polypeptide that carries out two separate reactions (1) the dehydration of citrate to form cis-aconitate and then (2) the hydration of ds-aconitate to yield isocitrate (see Figure 8-9). 

Reaction 2. The enzyme aconitase catalyzes the dehydration of citrate, producing ds-aconitate. The same enzyme, aconitase, then catalyzes addition of a water molecule to the ds-aconitate, converting it to isocitrate. The net effect of these two steps is the isomerization of citrate to isocitrate ... 

A wide range of metal ions is present in metalloenzymes as cofactors. Copper zinc snperoxide dismntase is a metalloenzyme that nses copper and zinc to help catalyze the conversion of snperoxide anion to molecnlar oxygen and hydrogen peroxide. Thermolysin is a protease that nses a tightly bonnd zinc ion to activate a water atom, which then attacks a peptide bond. Aconitase is one of the enzymes of the citric acid cycle it contains several iron atoms bonnd in the form of iron-sulfur clusters, which participate directly in the isomerization of citrate to isocitrate. Other metal ions fonnd as cofactors in metalloenzymes include molybdenum (in nitrate rednctase), seleninm (in glutathione peroxidase), nickel (in urease), and vanadinm (in fungal chloroperoxidase). see also Catalysis and Catalysts Coenzymes Denaturation Enzymes Krebs Cycle. 

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). 

In leucine biosynthesis, the intermediate 218 on the valine pathway reacts with acetyl-CoA to yield a-isopropylmalate 225 whose configuration has been shown to be S by X-ray crystallography (200). This reaction is analogous to that catalyzed by citrate synthase, and indeed the subsequent reaction, dehydration/rehydration giving ) -isopropylmalate 226, is analogous to the conversion of citrate to isocitrate. The configuration of / -isopropylmalate 226 had been shown to be 2i ,3S (201), and so the stereochemistry of the citrate and isopropylmalate reactions was identical. j -Isopropylmalate 226 was finally converted to leucine 205 by a 4-pro-R NADH specific dehydrogenase (EC 1.1.1.85) (202) and transamination (Scheme 61). 

Step 2. Isomerization of Citrate to Isocitrate The second reaction of the citric acid cycle, the one catalyzed by aconitase, is the isomerization of citrate to isocitrate. The enzyme requires Fe +. One of the most interesting features of the reaction is that citrate, a symmetrical (achiral) compound, is converted to isocitrate, a chiral compound, a molecule that cannot be superimposed on its mirror image. 

It is often possible for a chiral compound to have several different isomers. Isocitrate has four possible isomers, but only one of the four is produced by this reaction. (We shall not discuss nomenclature of the isomers of isocitrate here. See Question 28 at the end of this chapter for a question about the other isomers.) Aconitase, the enzyme that catalyzes the conversion of citrate to isocitrate, can select one end of the citrate molecule in preference to the other. 

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