The Enzyme Aconitase

The X-ray structures of other aconitases have appeared in the literature. Recently, the crystal structure of human iron regulatory protein, IRPl, in its aconitase form, has been published. Iron regulatory proteins (IRPs) control the translation of proteins involved in iron uptake, storage, and utilization by binding to specific noncoding sequences of the corresponding mRNAs known 

TABLE 7.11 Selected 7ACN Bond Distances (PDB 7ACN) 

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The enzyme aconitase catalyzes the hydration of aconitic acid to two products citric acid and isocitric acid. Isocitric acid is optically active citric acid is not. What are the respective constitutions of citric acid and isocitric acid ... 

In the presence of the enzyme aconitase, the double bond of aconitic acid undergoes hydration. The reaction is reversible, and the following equilibrium is established ... 

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. 

In the EPR of mammalian cells, we do not see much in addition to the signals from the respiratory complexes. The enzyme aconitase from the citric-acid cycle can be detected, and also the protein cytoplasmic aconitase, later identified as the mRNA translation regulatory factor iron regulatory protein IRP-1, which actually started its career in biochemistry as an EPR signal that could not be assigned to the respiratory chain (Kennedy et al. 1992). 

Compared to conventional NMR isotopes (13C, 15N, 2H), 19F-labels cannot be readily placed into proteins in a versatile manner by any biosynthetic expression strategy. Certain auxotrophic bacterial strains can be used to incorporate iso-steric 19F-labelled amino acids (e.g. Fluoro-Phe, Fluoro-Trp, Fluoro-Leu, Fluoro-Ile see Fig. 3), but yields tend to be low. Many other fluoro-organics are toxic if they get converted into fluoroacetic acid, which blocks the enzyme aconitase in the citric... 

Iron-sulfur clusters, such as those found in the enzyme aconitase discussed in Section 7.9.2.1, cannot be treated using the 16-e or 18-e rules. Other frameworks exist to treat large metal clusters, and these have some utility in treating [Fe,cSj,]" clusters. One method treats the number of metal atoms and the metal-metal bonds in a cluster according to the following formula ... 

One large class of non-heme iron-containing biomolecules involves proteins and enzymes containing iron-sulfur clusters. Iron-sulfur clusters are described in Sections 1.7 (Bioorganometallic Chemistry) and 1.8 (Electron Transfer) as well as in Section 3.6 (Mossbauer Spectroscopy). See especially Table 3.2 and the descriptive examples discussed in Section 3.6.4. Iron-sulfur proteins include rubredoxins, ferrodoxins, and the enzymes aconitase and nitrogenase. The nitrogenase enzyme was the subject of Chapter 6 in the hrst edition of this text—see especially Section 6.3 for a discussion of iron-sulfur clusters. In this... 

The Enzyme Aconitase. The enzyme aconitase catalyzes the elimination or addition of water in the second step of the citric acid (Krebs) cycle, catalyzing the interconversion of citrate and isocitrate via cix-aconitate. See reference 8, pages 190-196, Figure 7.49, and equation 7.13. 

Figure 7.49 Formation of isocitrate from citrate as catalyzed by the enzyme aconitase.

Citric acid has three prochiral centres The Krebs cycle is a process involved in the metabolic degradation of carbohydrate (see Section 15.3). It is also called the ciU ic acid cycle, because citric acid was one of the first intermediates identified. Once formed, citric acid is modified by the enzyme aconitase through the intermediate... 

This naturally occurring toxicant is an analogue of acetate and is incorporated into acetyl CoA (fluoroacetate) and hence into Krebs cycle (TCA cycle) as fluorocitrate. This blocks the enzyme aconitase, as the fluorine atom cannot be removed. The TCA cycle is blocked, and citrate accumulates. The mitochondrial energy supply is disrupted, hence cardiac damage occurs. Lack of oxaloacetate will allow ammonia to accumulate leading to convulsions. 

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... 

Answer Oxidation of co-fluorooleate in the /3-oxidation pathway forms fluoroacetyl-CoA in the last pass through the sequence. Entry of fluoroacetyl-CoA into the citric acid cycle produces fluorocitrate, a powerful inhibitor of the enzyme aconitase. As a result of this inhibition, the citric acid cycle shuts down and the flow of reducing equivalents to oxidative phosphorylation is fatally impaired. 

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

While a pro-chiral molecule is achiral, its two equivalent groups can be distinguished if it interacts with a chiral receptor. This was noted by Alexander Ogston, who explained why the two identical -CH2-COO groups in pro-chiral citrate are distinguished by the enzyme aconitase. One -CH2-COO group of citrate is derived from oxaloacetate, while the... 

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). 

Biotransformation, especially phase I metabolic reactions, cannot be assumed to be synonymous with detoxification because some drugs (although a minority) and xenobiotics are converted to potentially toxic metabolites (e.g. parathion, fluorine-containing volatile anaesthetics) or chemically reactive intermediates that produce toxicity (e.g. paracetamol in cats). The term lethal synthesis refers to the biochemical process whereby a non-toxic substance is metabolically converted to a toxic form. The poisonous plant Dichapetalum cymosum contains monofluoroacetate which, following gastrointestinal absorption, enters the tricarboxylic acid (Krebs) cycle in which it becomes converted to monofluorocitrate. The latter compound causes toxicity in animals due to irreversible inhibition of the enzyme aconitase. The selective toxicity of flucytosine for susceptible yeasts (Cryptococcus neoformans, Candida spp.) is attributable to its conversion (deamination) to 5-fluorouracil, which is incorporated into messenger RNA. 

The second metabolic pathway which we have chosen to describe is the tricarboxylic acid cycle, often referred to as the Krebs cycle. This represents the biochemical hub of intermediary metabolism, not only in the oxidative catabolism of carbohydrates, lipids, and amino acids in aerobic eukaryotes and prokaryotes, but also as a source of numerous biosynthetic precursors. Pyruvate, formed in the cytosol by glycolysis, is transported into the matrix of the mitochondria where it is converted to acetyl CoA by the multi-enzyme complex, pyruvate dehydrogenase. Acetyl CoA is also produced by the mitochondrial S-oxidation of fatty acids and by the oxidative metabolism of a number of amino acids. The first reaction of the cycle (Figure 5.12) involves the condensation of acetyl Co and oxaloacetate to form citrate (1), a Claisen ester condensation. Citrate is then converted to the more easily oxidised secondary alcohol, isocitrate (2), by the iron-sulfur centre of the enzyme aconitase (described in Chapter 13). This reaction involves successive dehydration of citrate, producing enzyme-bound cis-aconitate, followed by rehydration, to give isocitrate. In this reaction, the enzyme distinguishes between the two external carboxyl groups... 

Recent work has been directed toward exploring the behavior of the enzyme aconitase 31-33), Presently, CRAMS is being used to obtain an insight into the mechanisms of reactions involving this enzyme In this part, the problem is first stated in general terms, then a step by step description of the use of CRAMS is given. 

It is known that the enzyme aconitase catalyzes (1) the dehydration of both citrate and isocitrate to form cis-aconitate, (2) the reverse reactions, and (3) the interconversion of citrate and isocitrate thusly ... 

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