Aconitate isocitric acid

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 ... 

An attempt was made to convert aconitic acid (2 g) to isocitric acid using 25 ml. of 30% aqueous hydrogen peroxide, time and temperature unspecified. A crystalline product precipitated which exploded on grinding in a mortar. 

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. 

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. 

Fig. 3. Krebs citric acid cycle. Enzymes involved (1) Condensing enzyme (2) aconitase (3) isocitric acid (4) a-ketoglucaric acid dehydrogenase (4) a succinic acid thiokinasc (5) succinic acid dehydrogenase (6) fumarasc (7) malaic acid dehydrogenase. Abbreviations CA = citric acid ACOM = eij-aconitic acid KG = a-ketoglutaric acid SIC = succinic acid FA = fumaric acid MA = malic acid OA = oxalaceiic acid...

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... 

C volume of fen.-pentanol added, 200 ml flow velocity, 27 ml/h. Peaks 1.1—1.3 = ketoglutaric acid 2 = ew-aconitic acid 3 = malic acid 4 = citric acid 5 < isocitric acid. (From ref. 45 with permission of Preston Technical Abstracts Co., Niles, 111.)... 

Chiral (isocitric acid) Aconitic acid Achiral (citric add)... 

This conclusion is summarized in Fig. 64 which shows that, given the known configuration of isocitric acid U1) (2jR,3S), the addition of water to aconitic acid to give either citric or isocitric acid proceeds in antiperiplanar fashion to the Re face at C(2) and the Re face at C(3) in m-aconitic acid. And, finally, the addition of acetyl-CoA to oxaloacetic acid proceeds from the Si side of the carbonyl function.34... 

Although citrate has been excluded as the primary condensation product of pyruvate and oxalacetate, no direct evidence bearing upon the nature of this product has as yet been obtained. The participation of cfs-aconitic and isocitric acids is speculative. Nor is there any evidence supporting the hypothesis that pyruvate and oxalacetate condense to form a hypothetical intermediate oxalcitraconic acid which can be oxidatively decarboxylated to citric acid. Since citrate, aconitate and isocitrate are in equilibrium with each other, the participation of the last two substances as intermediates of carbohydrate oxidation would, on the surface, appear to be doubtful. Krebs, however, believes that the conversion of cis-aconitate to a-ketoglutarate occurs so rapidly in liver that equilibrium with citrate is not attained. 

Citric acid is then converted to its isomer, isocitric acid, by way of the enzyme aconitaset which functions, in effect, by removing a molecule of water from the citric acid (producing aconitic acid, which, however, remains bound to the enzyme and is not released into the cell solution at all) and then replacing the water molecule isomerically (18) ... 

Other C-0 lyase enzymes include aconitate hydratase or aconitase (E. C. 4.2.1.3), an enzyme that catalyzes two tricarboxylic acid cycle steps from isocitric acid to citrate (14)1141 or vice versa, via the intermediate cis-aconitate (13). Citrate dehydratase (E. C. 4.2.1.4) is only capable of converting citrate to cis-aconitate and does not act on isocitrate (15) 115l... 

Citric acid is isomerized by aconitase to yield isocitric acid (cw-aconitic acid is an intermediate). The oxidation of isocitrate by isocitrate dehydrogenase to a-oxoglutarate involves the formation of NADH (an NADP linked enzyme is also found) and the oxalosuccinic acid produced is de-carboxylated to yield a-oxoglutarate. 

Aliphatic tricarboxylic acids such as citric acid, isocitric acid, tricarballylic acid, and aconitic acid exhibit a remarkably high affinity toward the stationary phase of carbonate-selective anion exchangers. Hence, low-ionic strength car-bonate/bicarbonate buffer solutions are not particularly suitable as eluents. However, when a sodium hydroxide solution at a comparatively high concentration (c 0.08 mol/L) is used, tricarboxylic acids may be eluted. When the detection of these compounds is carried out via electrical conductivity, a high-capacity suppressor system such as micromembrane (MMS) or self-regenerating suppressors must be used to reduce background conductivity. 

Among the eight enzymes used in the citric acid cycle of more or less comon organic reactions needed to convert the acetyl (CH3CO) group of acetyl coenzyme A (acetyl-CoA e.g., see this chapter and Chapter 11) into two molecules of carbon dioxide (CO2) is that one called aconitase. Aconitase has been carefully examined and it has been determined that, as shown in Table 8.7, the enzyme catalyzes the reversible isomerization of citric and isocitric acids through the intermediacy of d5-aconitic acid. 

Citric acid oy-aconitic acid L-isocitric acid... 

Aconitic acid involved in the TCA and glyoxylate cycles and the acid commonly occurring in nature has the c/5-configuration. The trans- om x has also been isolated from some plant materials - for example, sugarcane Saccharum offi-cinarum) juice (17), tomato (Lycopersicon esculentum) (56) or moss (Bryophyta) (34). However, some of the occurrences might be artifacts of the isolation procedures, for an interconversion between two isomers of aconitic acid has been reported (14, 81). In both cycles, cw-aconitic acid is formed upon dehydration of citric acid catalyzed by aconitase (aconitate hydratase) which also catalyzes the rehydration of cw-aconitate to isocitric acid. 

Of the four stereoisomers, erythro-Ti - and Ls-, and threo-T> - and Lg-isodtric acid, it is the t/j/ieo-Dg-isomer that occurs in nature. The formation of isocitric acid from c/s-aconitic acid is catalyzed by aconitase. This acid is also accumulated in Crassulacean plants as a result of dark CO2 fixation. 

Krebs H A, Eggleston L V 1944 Microdetermination of isocitric acid and cw-aconitic acids in biological material. Biochem J 38 426-437... 

L-Malic and citric acids are the major organic acids of fruits (Table 18.13). Malic acid is predominant in pomme and stone fruits, while citric acid is most abundant in berries, citrus and tropical fruits. (2R 3R)-Tartaric acid occurs only in grapes. Many other acids, including the acids in the citric acid cycle, occur only in low amounts. Examples are cis-aconitic, succinic, pyruvic, citramalic, fumaric, glyceric, glycolic, glyoxylic, isocitric, lactic, oxalacetic, oxalic and 2-oxoglutaric acids. In fruit juices, the ratio of citric acid to isocitric acid (examples in Table 18.14) serves as an indicator of dilution with an aqueous solution of citric acid. 

This is an enzyme occurring in many animal and plant tissues that accelerates the conversion of citric acids (1) into aconitic acid, and (2) then into isocitric acid. 

The three tricarboxylic acids, citrate, cfs-aconitate, and isocitrate, are always found together when they are formed in animal tissue, and it was therefore difficult to decide in which order they are formed. For reasons which cannot be discussed in full the order citrate —> as-aconitate — isocitrate was proposed originally. But it was always clear that this order was somewhat arbitrary. 

Ogston has recently put forward an idea which removes the difficulty of ascribing both reactions to one enzyme. It is a development of his theory discussed above of a three-point combination between enzyme and substrate. As already explained, the fumarate molecule in order to yield optically active malic acid must be so placed on the enzyme surface that only one double-bond component can react, but no direction need be exerted on the water in which H and OH are distributed. If it is now assumed that aconitase is constructed analogously to fumarase in that again no direction is exerted on the elements of water when they combine with aconitic acid, it is seen at once that two different compounds arise, namely, citric and isocitric acids. The occurrence of the reverse reaction would follow from the requirement of catalytic reversibility. 

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