Isocitric acid dehydrogenase

Also, the carboxylation of oxoglutaric acid to form isocitric acid catalyzed by isocitric acid dehydrogenase (ICDH) can be driven electrochemically using methyl viologen as mediator without reduced pyridine nucleotides as cofactors. Thus, electrolysis at — 0.95 V vs SCE of a 0.2 M tris buffer solution (pH 7.7)... 

Fig. 20. Electroenzymatic carboxylation of oxoglutaric acid with isocitric acid dehydrogenase (ICDH) using ferredoxin (Fd) as mediator and poly-L-lysine as promoter... 

The enzyme isocitrate dehydrogenase is one of the enzymes of the Krebs or citric acid cycle, a major feature in carbohydrate metabolism (see Section 15.3). This enzyme has two functions, the major one being the dehydrogenation (oxidation) of the secondary alcohol group in isocitric acid to a ketone, forming oxalosuccinic acid. This requires the cofactor NAD+ (see Section 11.2). For convenience, we are showing non-ionized acids here, e.g. isocitric acid, rather than anions, e.g. isocitrate. 

The second function, and the one pertinent to this section, is the decarboxylation of oxalosuccinic acid to 2-oxoglutaric acid. This is simply a biochemical example of the ready decarboxylation of a P-ketoacid, involving an intramolecular hydrogen-bonded system. This reaction could occur chemically without an enzyme, but it is known that isocitric acid, the product of the dehydrogenation, is still bound to the enzyme isocitrate dehydrogenase when decarboxylation occurs. 

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...

Fluorimetric methods involving six dehydrogenases have been used to determine the concentrations of 21 of the more common organic acids (58). Sensitivity of the methods range from 0.02 fxg/m for determination of D-isocitric acid with isocitrate dehydrogenase to 200 /xg/ml for determination of D-tartaric acid with malate dehydrogenase. 

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. 

In 1948 Ochoa demonstrated the existence of an enzyme, isocitric dehydrogenase, which catalyzed the oxidation of isocitric acid and required NADP. He was, however, unable to demonstrate the formation of the expected product oxalosuccinic acid (Fig. 1). The existence of this acid as an intermediate in the Krebs cycle had been postulated by Carl Martius. Ochoa was able to prepare the compound by chemical synthesis and showed that cell extracts catalyzed the decarboxylation of this very unstable /S-keto acid to a-ketoglutaric acid. Similar results were simultaneously obtained by Lynen in Germany. 

A fixation of CO similar to the one which takes place in the malic enzyme reaction occurs in the synthesis of isocitric acid. The isocitric dehydrogenase system was shown to catalyze the reversible reaction (30) (2.59). Synthesis of d-isocitric acid, like the... 

Isocitric acid can supply hydrogen to NAD or NADP. Whereas NAD-isocitrate dehydrogenase was found exclusively in mitochondria, 80% of NADP-isocitrate dehydrogenase was localized in cytoplasm and only 20% in mitochondria (Ballard and Hanson, 1967). 

Inherently, the decarboxylation of p-keto acids and malonic acids (1) proceeds very smoothly, as the resulting product bearing anion adjacent to carbonyl group stabilizes as its enolate form (2) [Eq. (1)]. Enzyme-mediated reaction sometimes utilizes this facilitated decarboxylation. Indeed, isocitric acid (3) was oxidized to the corresponding keto acid, which subsequently decarboxylated to a-ketoglutaiic acid (4) by means of isocitrate dehydrogenase (EC 1.1.1.41) [Eq. (2)]. Another example is observed in the formation of acetoacetyl-CoA (5), which occupies the first step of fatty acid biosynthesis. A p-keto carboxylate 6, derived from the acetylation of malonyl-CoA with acetyl-CoA, decarbox-ylates to 5 by the action of 3-ketoacyl synthase [Eq. (3)]. 

Step 3 of Figure 29.12 Oxidation and Decarboxylation (2K,3S)-lsocitrate, a secondary alcohol, is oxidized by NAD+ in step 3 to give the ketone oxalosuccinate, which loses C02 to givea-ketoglutarate. Catalyzed by isocitrate dehydrogenase, the decarboxylation is a typical reaction of a /3-keto acid, just like that in the acetoacetic ester synthesis (Section 22.7). The enzyme requires a divalent cation as cofactor, presumably to polarize the ketone carbonyl group. 

Figure 22-4. Sequence of reactions in the oxidation of unsaturated fatty acids, eg, linoleic acid. A -c/s-fatty acids or fatty acids forming A -c/s-enoyl-CoA enter the pathway at the position shown. NADPH for the dienoyl-CoA reductase step is supplied by intramitochondrial sources such as glutamate dehydrogenase, isocitrate dehydrogenase,and NAD(P)H transhydrogenase.

Several mutant strains of R. eutropha that were made to possess defective competing metabolic pathways with the PHA biosynthetic pathway were developed for the enhanced PHA production. The isocitrate dehydrogenase leaky mutant of R. eutropha accumulated P(3HB) more favorably at a lower car-bon/nitrogen molar ratio and at a lower carbon concentration than the parent strain [82]. In batch culture, the final cell and P(3HB) concentrations, and P(3HB) yield on glucose were slightly increased. Also, in the P(3HB-co-3HV) biosynthesis, the molar fraction of 3HV and the 3HV yield on propionic acid increased due to the enhanced conversion of propionic acid to 3-hydroxyvaleryl-CoA rather than to acetyl-CoA and C02 in this mutant. Another mutant R. eu-... 

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