Aconitase, reaction catalyzed

Wachtershanser has also suggested that early metabolic processes first occurred on the surface of pyrite and other related mineral materials. The iron-sulfur chemistry that prevailed on these mineral surfaces may have influenced the evolution of the iron-sulfur proteins that control and catalyze many reactions in modern pathways (including the succinate dehydrogenase and aconitase reactions of the TCA cycle). 

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. 

Citrate rearranges to isocitrate in a reaction catalyzed by aconitase. 

FIGURE 16.2. Comparison of citrate and isocitrate (Ref. 2) with respect to the reaction catalyzed by the enzyme aconitase. The asterisk shows the hydrogen atom abstracted by the enzyme. The arrows indicate possible sites of binding to the enzyme (via carboxyl groups of the anions). 

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 ... 

The enzyme aconitase catalyzes the isomerization of citrate into isocitrate. Discuss the two reactions catalyzed by aconitase in terms of the chemistry of alcohols and alkenes. 

Cells readily convert fluoroacetate to fluoroacetyl-CoA in a reaction catalyzed by the enzyme acetate thiokinase (reaction diagram). Fluoroacetyl-CoA can combine with oxaloacetate to form fluorocitrate in a reaction catalyzed by the citric acid cycle enzyme, citrate synthase. Fluorocitrate is toxic to cells because it inhibits aconitase. 

Recall With respect to stereochemistry, what is unique about the reaction catalyzed by aconitase ... 

Could the citric acid cycle proceed under standard conditions Why or why not Given the thermodynamic data you have gathered about the reactions catalyzed by aconitase, how can the citric acid cycle proceed under cellular conditions ... 

Little was known of the mechanisms of action of aconitase because all attempts at purification failed until it was demonstrated that the enzyme requires iron and cysteine for activity. It has since become evident that the mechanism of the reaction catalyzed by aconitase is extremely complex. C/.y-aconitate was thought to be an intermediate, but the compound could not be isolated. This led Speyer and Dickman [78] to suggest that a common intermediate exists between citrate on the one hand, and aconitate and isocitrate on the other (see Fig. 1-17). These authors found that when citric acid labeled with heavy water was used as a substrate, the isocitrate was extensively labeled, while only traces of deuterium were found in cw-aconitate. This suggested that cw-aconitate is not on the pathway leading from isocitric to citric acid. To explain these results, the authors postulated an intermediate common to c/.y-aconitate and isocitrate consisting of a tricarboxylic acid forming a complex with iron and cysteine. Such a complex would then be capable of intramolecular hydrogen transfer between the carbonium... 

Like aconitase, fumarase catalyzes a reversible hydration-dehydration reaction. The fumarase mani-... 

Equilibria of Aconitase and Fumarase. The reactions catalyzed by fumarase and aconitase have small free-energy changes. Omitting water from the calculation, the equilibrium constant for fumarase, K, = (malate)/(fumarate) = about 3-4 at room temperature and pH 7. This reaction is temperature dependent, forming relatively more fumarate at higher temperatures. The aconitase equilibrium must include two separate reactions the equilibration of cfs-aconitate with... 

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

In contrast to laboratory reactions, enzyme-catalyzed reactions often give a single enantiomer of a chiral product, even when the substrate is achiral. One step in the citric acid cycle of food metabolism, for instance, is the aconitase-catalyzed addition of water to (Z)-aconitate (usually called ris-aconitate) to give isocitrate. 

Problem 9.26 The aconitase-catalyzed addition of water to ds-aconitate in the citric acid cycle occurs with the following stereochemistry. Does the addition of the OH group occur on the Re or the Si face of the substrate What about the addition of the H Does the reaction have syn or anti stereochemistry ... 

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. 

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.

There have been a number of isolated studies of metal-ion catalyzed nucleophilic reactions of other groupings. Particularly interesting is the induced nucleophilic attack on olefins. Hydration is normally very sluggish. Enzymes can speed up such reactions. Aconitase, an iron-containing enzyme, catalyzes the isomerization of citric acid to isocitric acid, through the intermediacy of cis-aconitic acid. A possible mechanism has been suggested based on the following Co(III) model chemistry. Rapid cyclization of the maleate ester produces Ai and AS chelated malate half ester ... 

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). 

Yeast isopropylmalate isomerase of the leucine biosynthetic pathway, which catalyzes a totally analogous reaction to that of aconitase, converts 3-hydroxy-3-carboxy-4-methylpentanoate to 2-hydroxy-3-carboxy-4-methylpentanoate via an allylic intermediate. In its initial characterization by EPR spectroscopy, a high-field shift in its EPR signal from a g-average of 1.96 to 1.90 is seen upon addition of substrate (70). This result suggests that its mechanism is the same as that found for aconitase. 

Enzymes usually function stereospedfically. In chiral substrates, they only accept one of the enantiomers, and the reaction products are usually also sterically uniform. Aconitate hydratase (aconitase) catalyzes the conversion of citric acid into the constitution isomer isocitric acid (see p.l36). Although citric acid is not chiral, aconitase only forms one of the four possible isomeric forms of isocitric acid (2i ,3S-isocitric acid). The intermediate of the reaction, the unsaturated tricarboxylic acid aconitate, only occurs in the cis form in the reaction. The trans form of aconitate is found as a constituent of certain plants. 

A somewhat different type of reaction is catalyzed by the iron (II) -containing (12) enzyme aconitase, which brings about the interconversion of citric, isocitric, and aconitic acids by hydration and dehydration. 

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. 

The enzyme aconitase. which contains the Fe2+ ion at the reactive center, catalyzes the interconversion of citric, isocilric, and aconilic acids. The reaction has been shown to occur through the formation of a single intermediate carbonium ion structure in which the Fc2+ ion is always bound to the same donor aloms. while the interconversion of the substrate occurs through the migration of only protons and electrons. 

The enzyme aconitase catalyzes the isomerization of citric acid to isocitric acid via the intermediate cis-aconitic acid (Scheme 46),530 and various attempts have been made to model this reaction.21 The cobalt Ill) complexes derived from methyl maleate (171) and methyl fumarate (172) have been prepared531 to study intramolecular attack by coordinated hydroxide on the alkene. Generation of the hydroxo species of the maleic acid complex leads to rapid cyclization to give the... 

Hydration and/or dehydration reactions are frequently catalyzed by metallopro-teins. Examples are proteins containing nickel (urease), zinc (e.g., peptidases), molybdenum (the hydratase partial reaction of formate oxidoreductase), tungsten (acetylene hydratase). An obvious difference between Ni, Zn, on the one hand, and Fe, Mo, W, on the other, is that the first are directly coordinated to the protein whereas the latter are also part of a cofactor. With reference to the Fe/S cluster in aconitase it has been suggested that cofactor coordination may provide an added flexibility to the active site, in particular to the substrate binding domain [15],... 

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],... 

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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