Action of Aconitase

The foregoing results lead to the inescapable conclusion that substrate binding is a subsite-specific event. The species along the reaction pathway that are responsible for doublets 5, and S2 have not been unambiguously identified (128). The O and ENDOR spectroscopic findings by Hoffman and co-workers (164, 165), however, using isotopically labeled substrate, intermediate, and inhibitors have been more informative in this 

Reorientation of ciy-aconitate such that the previously free carboxylate group becomes coordinated, followed by the stereospecific events of hydroxide delivery to the activated olefinic carbon atom and protonation at the other carbon atom, generates the isocitrate product. 

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Iron appears to play a significant role in the mechanism of action of aconitase, a constituent enzyme of the tricarboxylic acid cycle (32). 

The first attempt to determine the configuration of citrate was that of Martius and Schorre [106,107]. At the time of their work (1950) it was reasonably clear that the action of aconitase was on that portion of the citrate molecule derived from oxaloacetate. This follows from the following observations. 

These experiments can be interpreted in two ways, depending on the configuration of citrate. However, in each case, the action of aconitase must be such as to lead to introduction of the double bond of m-aconitic acid between the carbons originally derived from oxaloacetate. This follows from the fact that the carbonyl... 

The NADPH was isolated after the subsequent action of aconitase and isocitrate dehydrogenase on the two citrate samples. 

If [2,3-2H2]fumarate is used as substrate for the fumarase reaction in HzO, the malic acid has (5) configuration at C-3, 123 in this case, the proton lost by action of aconitase is H not 2H, and 2H is finally removed by action of isocitrate dehydrogenase. Using normal projection formulae, this sequence is as follows ... 

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... 

The equations usually written to describe the action of aconitase show cffi-aconitate as an intermediate between citrate and isocitrate. Experiments with deuterated water have recently indicated that a more correct formulation is the interconversion of all of the three tricarboxylic acids through a common intermediate (I). The most convincing experiments... 

It is noteworthy that except for the Rieske center in Complex III, Complexes I and 11 are home to all the iron-sulfur clusters in the mitochondrial electron transfer chain and consequently most of the iron-containing carriers in the entire sequence. Hibbs subsequently showed that CAM-injured cells lose a substantial portion of their total intracellular iron (Hibbs et al., 1984) [later studies specifically identified loss of mitochondrial iron (Wharton et al., 1988)] and Drapier and Hibbs (1986) showed that the activity of another iron-sulfur-containing enzyme, aconitase, is also lost. In early 1987 Hibbs reported that the cytostatic actions of CAMs requires the presence of only one component in culture medium, L-arginine (Hibbs et al., 1987b). Thus, the stage was set for the discovery of a unique reactive species that targets intracellular iron, produced by CAMs. 

The citrate ion, a very important prochiral metabolic intermediate, has three prochiral centers at C-2, C-3, and C-4, respectively. That at C-3 distinguishes the pro-R and pro-S arms and determines the stereochemical numbering. Citrate containing 14C in the sn-1 position is called s -citrate[l-14C] and is the form of labeled citrate that is synthesized in living cells from oxaloacetate and [l-14C]acetyl coenzyme A (see Fig. 10-6). The first step in the further metabolism of citrate is the elimination of the -OH group from C-3 together with the Hr proton from C-4 through the action of the enzyme aconitate hydratase (aconitase). In this case the proton at C-4 (in the pro-R arm) is selected rather than that at C-2. 

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 ... 

The activity of an enzyme may be inhibited by the presence of a toxic metabolite. Sodium fluoroacetate, known as rat poison 1080, is extremely toxic to animals. The toxic action, however, is not due to sodium fluoroacetate itself but to a metabolic conversion product, flu-orocitrate, formed through a reaction commonly known as "lethal synthesis," as shown in Figure 5.3. The resulting fluorocitrate is toxic because it is inhibitory to aconitase, the enzyme responsible for the conversion of citrate into czs-aconitate and then into isocitrate in the tricarboxylic acid cycle. Inhibition of aconitase results in citrate accumulation. The cycle stops for lack of metabolites, leading to disruption of energy metabolism. 

Villafranca, J, J., Mildvan, A. S. The Mechanism of Aconitase Action, III. Detection and Properties of Enzyme — Metal — Substate and Enzyme — Metal — Inhibitor Bridge Complexes with Manganese (II) and Iron (II). J. Biol. Chem. 247, 3454 (1972). 

Villafranca, J. J. The Mechanism of Aconitase Action, IV. Inhibition of Aconitase by Tricarboxylic Acids (in preparation). 

D-Isocitrate is a citric acid cycle (and glyoxylate cycle) intermediate produced as a result of action of the enzyme aconitase on citrate. Isocitrate is converted to... 

Iron is also an essential constituent of several non-porphyrin enzymes, e.g. aconitase, aldolase, and succinic dehydrogenase. Inhibition of the synthesis of glucose by tryptophan in animal cells depends on chelation. The tryptophan is metabolized to pyridine-2,3-dicarboxylic acid, which complexes the divalent iron necessary for the action of phosphoenolpyruvate carboxykinase (a key enzyme in the neogenesis of glucose) (Veneziale et al., 1967). 

Whereas AZ-bromoacetamide inhibits the enzymic action of rennin, dipotassium ( -aconitate (178) is of interest since it is a substrate of aconitase. The di-ionized draivative (178) proves to be almost exactly planar, and contains a short intramolecular O-H - O hydrogen bond of 2.425 A which probably serves to stabilize the conformation. It is suggested... 

Hence, it is not surprising, that all of the eight intermediates in the TCA cycle earned a lot of attention in modem life sciences. Although a naive approach may be to assume the TCA cycle to be a finished chapter in the book of science, a closer look reveals this statement to be misleading. One member managed to escape at least the chemists access until today ICA. It is formed by action of the enzyme aconitase from CA via the intermediate c/ -aconitate (c/5 -aconitic acid [499-12-7]). 

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