Aconitate citrate

L-glutamic acid C. melassecola C. melassecola Glu A, citrate dehydrogenase, ppc, aconitate dehydratase ... 

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... 

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). 

Inspection of the citrate structure shows a total of four chemically equivalent hydrogens, but only one of these—the pro-/J H atom of the pro-i arm of citrate—is abstracted by aeonitase, which is quite stereospecific. Formation of the double bond of aconitate following proton abstraction requires departure of hydroxide ion from the C-3 position. Hydroxide is a relatively poor leaving group, and its departure is facilitated in the aeonitase reaction by coordination with an iron atom in an iron-sulfur cluster. 

Elucidating the stereochemistry of reaction at prochirality centers is a powerful method for studying detailed mechanisms in biochemical reactions. As just one example, the conversion of citrate to (ds)-aconitate in the citric acid cycle has been shown to occur with loss of a pro-R hydrogen, implying that the reaction takes place by an anti elimination mechanism. That is, the OH and H groups leave from opposite sides of the molecule. 

The dehydration of citrate to yield c/s-aconitate, a step jn the citric acid cycle, involves the pro-R "arm" of citrate rather than the pio-S arm. Which of the following two products is formed ... 

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. 

Problem 29.11 Write mechanisms for step 2 of the citric acid cycle, the dehj dration of citrate and the addition of water to aconitate. 

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. 

Figure 3 Gradient separation of anions using suppressed conductivity detection. Column 0.4 x 15 cm AS5A, 5 p latex-coated resin (Dionex). Eluent 750 pM NaOH, 0-5 min., then to 85 mM NaOH in 30 min. Flow 1 ml/min. 1 fluoride, 2 a-hydrox-ybutyrate, 3 acetate, 4 glycolate, 5 butyrate, 6 gluconate, 7 a-hydroxyvalerate, 8 formate, 9 valerate, 10 pyruvate, 11 monochloroacetate, 12 bromate, 13 chloride, 14 galacturonate, 15 nitrite, 16 glucuronate, 17 dichloroacetate, 18 trifluoroacetate, 19 phosphite, 20 selenite, 21 bromide, 22 nitrate, 23 sulfate, 24 oxalate, 25 selenate, 26 a-ketoglutarate, 27 fumarate, 28 phthalate, 29 oxalacetate, 30 phosphate, 31 arsenate, 32 chromate, 33 citrate, 34 isocitrate, 35 ds-aconitate, 36 trans-aconitate. (Reproduced with permission of Elsevier Science from Rocklin, R. D., Pohl, C. A., and Schibler, J. A., /. Chromatogr., 411, 107, 1987.)...

Exudation occurs in response to environmental constraints, especially P deficiency (e.g., Jones 1998 Hinsinger et al. 2003) and differs depending on the P-form (Lambers et al. 2002) and plant species (Nuruzzaman et al. 2006). Banksia grandis exuded citrate, malate, and trans-aconitate when supplied with aluminium-phosphate. It exuded less of these tricarboxylates and dicarboxylates, but instead lactate and acetate, when supplied with iron-phosphate (Lambers et al. 2002). Plant species differ in their abilities to use various P species (van Ray and van Diest 1979), which can be due to differences in their exudation behavior (Nuruzzaman et al. 2006) and acidification of the root zone (Haynes 1992). This can influence the interspecific competition and coexistence of species, as we will discuss later. 

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 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. 

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. 

Gawron et al. (13,14) determined the stereochemistry of natural isocitric acid by chemical means. The results require the rrons-addition of water across the cis-aconitate intermediate double bond to produce either citrate or 2R,3S-isodtrate. Mass and NMR analyses of isotopically labeled citrate and isocitrate in the early 1960 s (15-17), defined the stercospedficities of the dehydration steps. These results led Gawron to propose the binding of cis-aconitate to the active site in two orientations differing by a 180° rotation about the double bond, as shown in Equation 2. This allows for the protonation by a base (-BH) and hydroxylation of the double bond to occur on aconitase at single, separate loci for the formation of either citrate or isocitrate. 

Figure 7. EPR absorption derivative spectra of photoreduced active aconitase. Enzyme ( 5 mg/ml) in 100 mM Hepes, pH 7.5, plus 5 pM deazaflavin and 10 mM potassium oxalate was photoreduced in the presence of either A) 10 mM tricarb-allylate, B) 1 mM citrate, or C) 10 mM rrans-aconitate. The numbers above each spectrum are the g-values of prominent features. Experimental conditions for obtaining EPR spectra were 13 K, 1 milliwatt microwave power, 0.8 mT modulation amplitude, and 9.24 GHz microwave frequency.

The details obtained fiom the ENDOR work, in addition to past results, allow for the following mechanism for aconitase. In Scheme II the R group on the substrate is CH2COO, -B represents the amino acid side-chain which stereospeci-fically transfers a proton between citrate and isocitrate, and X is either water or a protein ligand. In the presence of c/s-aconitate, bound water can freely exchange... 

Citrate is subsequently isomerized to isocitrate this involves dehydration and rehydration via the intermediate cis-aconitate. Both reactions are... 

This [4Fe-4S] cluster-containing enzyme [EC 4.2.1.3], also known as citrate hydro-lyase and aconitate hydra-tase, will act on citrate to generate ds-aconitate ((Z)-prop-l-ene 1,2,3-tricarboxylate) and water. The enzyme will also catalyze the conversion of isocitrate into ds-aconitate. 

The toxicity of fluoroacetic acid and of its derivatives has played an historical decisive role at the conceptual level. Indeed, it demonstrates that a fluorinated analogue of a natural substrate could have an activity profile that is far different from that of the nonfluorinated parent compound. The toxicity of fluoroacetic acid is due to its ability to block the citric acid cycle (Krebs cycle), which is an essential process of the respiratory chain. The fluoroacetate is transformed in vivo into 2-fluorocitrate by the citrate synthase. It is generally admitted that aconitase (the enzyme that performs the following step of the Krebs cycle) is inhibited by 2-fluorocitrate the formation of aconitate through elimination of the water molecule is a priori impossible from this substrate analogue (Figure 7.1). 

Although this interpretation can be found in most books, the reality is much more complex. Citrate synthase yields the sole (2/f,3/f)-2-fluorocitrate with a large stereoselectivity. This stereomer (and three other ones) does not inhibit aconitase, but it is a substrate of this enzyme. Aconitase cannot afford cw-aconitate by elimination since... 

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