Cis -Aconitate

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). 

Aliphatic acids Formic, acetic, butyric, popionic, malic, citric, isocitric, oxalic, fumaric, malonic, succinic, maleic, tartaric, oxaloacetic, pyruvic, oxoglutaric, maleic, glycolic, shikimic, cis-aconitic, trans-aconitic, valeric, gluconic... 

Acid yellow (Aquashade), herbicide/algicide for aquaculture in U.S., 3 214t Acitretin, 25 790 a-c Josephson effect, 15 750 Aclame, 24 232 ac losses, 23 845-846 Acne, vitamin A treatments for, 25 789 cis-Aconitic acid, in citric acid cycle, 6 633 Aconitine, 2 103... 

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... 

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 ... 

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. 

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

This enzyme [EC 4.1.1.6] catalyzes the conversion of cis-aconitate to itaconate (or, 2-methylenesuccinate) and carbon dioxide. 

Figure 1. Total ion current of n-butyl esters of the acidic fraction from non-sterile leaf washings of C. sativa. Key 1 = oxalic acid 2 = malonic acid 3 = maleic acid 4 = succinic acid 5 = fumaric acid 6 = alpha-ketoglutaric acid 7 = cis-aconitic acid 8 = citric acid (19)-...

In contrast, selective inhibition of enzyme activity involves highly specific interactions between the protein and chemical groups on the xenobiotic. An excellent example of this type of inhibition is seen in the toxic effect of fluoroacetate, which is used as a rodenticide. Although fluoroacetate is not directly toxic, it is metabolized to fluoroacetyl-CoA, which enters the citric acid cycle due to its structural similarity to acetyl-CoA (Scheme 3.5). Within the cycle, fluoroacetyl-CoA combines with oxalo-acetate to form fluorocitrate, which inhibits the next enzyme, aconitase, in the cycle [42]. The enzyme is unable to catalyze the dehydration to cis-aconitate, as a consequence of the stronger C-F bond compared with the C-H bond. Therefore, fluorocitrate acts as a pseudosubstrate, which blocks the citric acid cycle and, subsequently, impairs ATP synthesis. 

Significant inhibition of all activities below pH 7 by citrate for which a Ki value of 5.2 mil/ has been observed at pH 6.5. Isocitrate much less effective, and only minor effects noted with cis-aconitate, f-malate, succinate, ar ketoglutarate, and oxalate. Malonate, tartrate, L-glutamate, glutarate, adipate, 0-hydroxy-0-methyl glutaryl-CoA, and 0-hydroxybutyrate also found ineffective as instantaneous inhibitors... 

Fig. 5.22. Oxidation of acetyl-CoA via the tricarboxylic acid (TCA) cycle. Individual enzymes of the pathway are marked. 1, citrate synthase 2 and 3, cis-aconitate hydratase 4 and 3, isocitrate dehydrogenase 6, a-oxo glutarate dehydrogenas 7, succinate thiokinase 8, succinate...

Oxalic, quinic, tartaric, malic, isocitric, ascorbic, citric, fumaric, succinic, propionic, cis-aconitic Orange juice YMC-Pack ODS-AQ (250 X 4.6 mm) 5 /ttm 20 mM KH2P04, pH 2.8 UV, 214 nm 116... 

In the late 1930s the tricarboxylic acid cycle (fig. 13.3) contained d.v-aconitate, but more modern versions of the cycle (since the late 1950s) do not. The involvement of cz.v-aconitate is still shown in many nonbiochemistry texts. Can you explain why cis-aconitate is deleted from modern versions of the tricarboxylic acid cycle ... 

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... 

In short, the reaction mechanism consists of a dehydration, a flip, and a hydration. The first and the last steps appear to be well defined on the basis of spectroscopy, crystallography, and chemical common sense. The details of the flip, perhaps the key feature of the mechanism, are less clear. There are no crystals of the cis-aconitate complex (in fact, there should be two different complexes, 5 and 7). The free-enzyme intermediate 6 has not been isolated. Displacement of one cis-aconitate by another one is expected to require significant conformational changes in the cleft from the catalytic center to the protein surface. It has not been established that the leaving and entering cA-aconitases are not, in fact, one and the... 

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