Isocitrate isolation

Schossberger has recently isolated the isomeric form of citral which is knowm as isocitral, corresponding to the isogeraniol resulting from enolised citral acetate. 

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 ... 

The enzyme has been isolated from many tissues, the best source being a heart muscle or yeast. The isocitrate dehydrogenase requires the presence of cofactors Mg2+ or Mn2+. As an allosteric enzyme, it is regulated by a number of modulators. ADP, adenosine diphosphate, is a positive modulator and therefore stimulates enzyme activity. The... 

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

A reactor containing a Dean-Stark trap was charged with isocitric acid lactone (14.3 mmol), propanediol (15.7 mmol), and benzene was then refluxed at 90°C overnight. The mixture was concentrated and a viscous liquid isolated that solidified on cooling. 

The majority of synthetic reactions in mammalian cells takes place in the cytosol. The intramitochondrial localization of transhydrogenase excludes a direct participation in these anabolic processes. Substrate shuttle mechanisms (176, 177) are required to allow for the interaction between intra- and extramitochondrial nicotinamide nucleotide-dependent reactions. In the first instance transhydrogenase can be regarded to be functionally related to intramitochondrial NADP-linked reactions. A number of studies on isolated mitochondria have elaborated these relationships in some detail, in particular with regard to mitochondrial monooxygenation reactions and to the metabolism of glutamate and isocitrate. 

X1B-X14), and NADP-linked isocitrate dehydrogenase SIS, S16). Numerous studies have been carried out with isolated mitochondria to identify the source of NADPH for 11 -hydroxylation by studying the effect of inhibitors and uncouplers of oxidative phosphorylation in the presence of different hydrogen donors (83, 19S, SIO, SIS, SI4-SSS). Part of this work was inconclusive since several complicating features in the metabolism of adrenocortical mitochondria were insufficiently taken into account such as secondary inhibitory effects of the substrates, inhibitors, or uncouplers used 83, SI4, SSO, SSS) the requirement of transport of substrates across the mitochondrial membrane SS3, SS4) and the possibility of intramitochondrial dismutation reactions SS3). 

Perhaps the best characterized example of a subsite differentiated [4Fe-4S] protein is aconitase, which catalyzes the citrate-isocitrate isomerization in the citric acid cycle (257). Aconitase isolated aerobically is inactive and contains a [3Fe-4S] cluster. Activity is restored by incubation with Fe and this also reconstitutes the [4Fe-4S] cluster. Oxidation of the core results in loss of the fourth iron atom, regenerating the [3Fe-4S] form. Mossbauer studies have demonstrated that only one of the four iron sites is exchanged (258). X-ray studies on both [3Fe-4S] and [4Fe-4S] forms of pig heart aconitase 258a) showed that insertion of iron into [3Fe-4S] occurs isomorphously. The positions of the common atoms in the two forms of the core agree to within 0.1 A, supporting the view of the [3Fe-4S] cluster as an iron-voided cubane. A similar result was obtained for the seven iron ferredoxin from Azo-... 

COs to form oxalacetate which under anaerobic conditions is reduced to malate. The malate in turn may be converted to fumarate and succinate (Fig, 5). The last step in this series of reactions is blocked by malonate. The second pathway involves the aerobic condensation of pyruvate and oxalacetate followed by oxidation of the condensation product to form -ketoglutarate and succinate. Wood has proposed that the first condensation product of the aerobic tricarboxylic cycle is cfs-aconitic acid which is then converted to succinate by way of isocitric, oxalosuccinic, and a-ketoglutaric acids. The a-ketoglutarate is decarboxylated and oxidized to succinic acid. Isotopic a-ketoglutarate containing isotopic carbon only in the carboxyl group located a to the carbonyl would be expected to yield non-isotopic succinate after decarboxylation. This accounts for the absence of isotopic carbon in succinate isolated from malonate-poisoned liver after incubation with pyruvate and isotopic bicarbonate. 

CJ-15,183 (44) has been isolated from the fermentation culture of the fungus, Aspergillus aculeatus CL38916 as a squalene synthase inhibitor. The compound potently inhibited rat liver and human squalene synthases. In addition, it showed antifungal activities against filamentous fungi and yeast. The structure was elucidated to be an aliphatic tetracarboxylic acid compound consisting of an alkyl y-lactone, malic acid and isocitric acid moieties by spectroscopic analyses [69]. 

The beef heart enzyme (M, = 80,000) (117) is a component of the citric acid cycle. Its active form contains one [4Fe-4S] cluster. Although such a cluster is normally associated with electron transfer, the enzyme catalyzes the nonredox reaction of citrate-isocitrate interconversion via a dehydration-hydration pathway. The current state of understanding of cluster structures and reactions of beef heart aconitase has been thoroughly reviewed by Emptage (130). When isolated aerobically, aconitase is inactive and contains one [3Fe-4S] cluster. Upon incubation of the reduced protein with Fe(ll), the fully active enzyme is generated. When a 3-Fe center is reduced to [3Fe-4S]°, Reaction 10 builds a 4-Fe cluster in a nonredox process. The Mossbauer spectra in Fig. 8 address the question of subsite specificity in this reaction of aconitase (124). If the externally supplied iron is Fe, the resultant spectrum reveals the intrinsic (original) Fe atoms... 

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