Cw-Aconitic acid

If aconitase is incubated with cw-aconitic acid (or either of the citrates) there is formed an equilibrium mixture containing 88.4% of citrate, 7.5% isocitrate and 4.1% of m-aconitate. It is possible that in the hydration-dehydration process... 

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. 

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. 

Coic Y, Lesaint Ch, Le Roux F 1961 The transformation of cw-aconitic acid to the trans isomer during chromatography on silica. CR Acad Sci 253 1124-1126... 

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

Aconitase is the trivial name for citrate dehydratase cw-aconitate hydratase (EC 4.2.1.3). It catalyzes the reversible isomerization reaction of citrate into isocitrate via the intermediate cA-aconitate (Figure 2). It is a water-soluble, monomeric protein. In eukaryotic cells aconitase is located in the mitochondrial matrix. In prokaryotes the enzyme occurs in the cytoplasma. The pig heart enzyme consists of 754 amino-acid residues, providing a molecular mass of 83 kDa [27], Aconitase from other sources has similar size. The porcine protein is synthesized with a mitochondrial targeting sequence. The mature, functional protein can be (over)expressed in Escherichia coli [28],... 

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... 

Fig. 3.2 Chromatogram of standard acids as their methyl esters, separated on a 3 ft (0.001 m i.d.) glass column packed with 5 per cent poly(ethylene glycol) adipate on silanized Gas Pack W (100-120 mesh) using temperature programming from 80°C to 180°C at 10°C min" with an initial isothermal delay of 1.33 min. Peak identifications are 1, lactate 2, pyruvate 3, malonate (internal standard) 4, fumarate 5, succinate 6, adipate (internal standard) 7, malate 8 and 9,2-oxoglutarate 10, cw-aconitate, 11, citrate 12, isocitrate. (Redrawn with modifications from De Silva, 1971)...

---