Oxalosuccinate. decarboxylation

Methylsuccinic acid has been prepared by the pyrolysis of tartaric acid from 1,2-dibromopropane or allyl halides by the action of potassium cyanide followed by hydrolysis by reduction of itaconic, citraconic, and mesaconic acids by hydrolysis of ketovalerolactonecarboxylic acid by decarboxylation of 1,1,2-propane tricarboxylic acid by oxidation of /3-methylcyclo-hexanone by fusion of gamboge with alkali by hydrog. nation and condensation of sodium lactate over nickel oxide from acetoacetic ester by successive alkylation with a methyl halide and a monohaloacetic ester by hydrolysis of oi-methyl-o -oxalosuccinic ester or a-methyl-a -acetosuccinic ester by action of hot, concentrated potassium hydroxide upon methyl-succinaldehyde dioxime from the ammonium salt of a-methyl-butyric acid by oxidation with. hydrogen peroxide from /9-methyllevulinic acid by oxidation with dilute nitric acid or hypobromite from /J-methyladipic acid and from the decomposition products of glyceric acid and pyruvic acid. The method described above is a modification of that of Higginbotham and Lapworth. ... 

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. 

Biotin is a growth factor for many bacteria, protozoa, plants, and probably all higher animals. In the absence of biotin, oxalacetate decarboxylation, oxalosuccinate carboxylation, a-ketoglutarate decarboxylation, malate decarboxylation, acetoacetate synthesis, citrulline synthesis, and purine and pyrimidine syntheses, are greatly depressed or absent in cells (Mil, Tl). All of these reactions require either the removal or fixation of carbon dioxide. Together with coenzyme A, biotin participates in carboxylations such as those in fatty acid and sterol syntheses. Active C02 is thought to be a carbonic acid derivative of biotin involved in these carboxylations (L10, W10). Biotin has also been involved in... 

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. 

It is appropriate here to look at the structure of oxaloacetic acid, a critical intermediate in the Krebs cycle, and to discover that it too is a P-ketoacid. In contrast to oxalosuccinic acid, it does not suffer decarboxylation in this enzyme-mediated cycle, but is used as the electrophile for an aldol reaction with acetyl-CoA (see Box 10.4). 

I. 1.1.42], also known as oxalosuccinate decarboxylase, catalyzes the reaction of isocitrate with NADP+ to produce a-ketoglutarate, carbon dioxide, and NADPH. The enzyme is reported to be able to decarboxylate added oxalosuccinate. 

The first oxidative conversion of the TCA cycle is catalyzed by isocitrate dehydrogenase. This conversion takes place in two steps oxidation of the secondary alcohol to a ketone (oxalosuccinate), followed by a j8 decarboxylation to produce a-ketoglutarate (fig. 13.9). 

NAD+ is the electron acceptor for the oxidative step, and Mg2+ or Mn2+ is required for the decarboxylation. The oxalosuccinate that is presumably an intermediate does not dissociate from the enzyme. 

The oxidative decarboxylation of isocitrate to a-kctoglutaratc, catalyzed by mitochondrial isocitrate dehydrogenase. The intermediate, oxalosuccinate, is not released from the enzyme. B represents a catalytic side chain from the enzyme. 

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. 

Some of the earliest kinetic studies on metal ion-promoted reactions were carried out on metal ion-promoted decarboxylations of j8-oxo acids. The literature on this topic up to about 1974 has been reviewed. Much of the work has centred on oxaloacetic acid (HO2CCOCH2CO2H = H20xac) and its derivatives, a,a-dimethyl oxaloacetic acid and fluorooxaloacetic acid. Studies have also been made with acetonedicarboxylic acid (3-oxoglutaric acid), " dihydroxyfumaric acid, dihydroxytartaric acid, acetosuccinic acid, oxalosuccinic acid and 2-oxalopropionic acid (Figure 6). The decarboxylation of )3-oxo acids is of considerable biological importance, and in a number of cases metalloenzymes are involved. Similarities in the enzymatic and chemical processes stimulated early interest in these reactions as models for the enzymatic systems. 

Isocitrate dehydrogenase catalyzes the first of two decarboxylations and dehydrogenations in the cycle. Three different isocitrate dehydrogenases are present one specific for NAD+ and found only in mitochondria, the other two specific for NADP+ and found in mitochondria and cytoplasm. The NAD -specific enzyme is the primary enzyme with regard to TCA cycle operation. All three require Mg + or Mn +. The reaction yields a-ketoglutarate (2-oxoglutarate), NAD(P)H, and CO2 and involves enzyme-bound oxalosuccinate as an intermediate. 

Isocitrate is oxidized to form NADH and C02. The oxidative decarboxylation of isocitrate, catalyzed by isocitrate dehydrogenase, occurs in two steps. First, isocitrate is oxidized to form oxalosuccinate, a transient intermediate ... 

Immediate decarboxylation of oxalosuccinate results in the formation of a-keto-glu-tarate, an a-keto acid. There are two forms of isocitrate dehydrogenase in mammals. The NAD+-requiring isozyme is found only within mitochondria. The other isozyme, which requires NADP+, is found in both the mitochondrial matrix and the cytoplasm. In some circumstances the latter enzyme is used within both compartments to generate NADPH, which is required in biosynthetic processes. Note that the NADH produced in the conversion of isocitrate to a-ketoglutarate is the first link between the citric acid cycle and the ETC and oxidative phosphorylation. 

A variety of metal ions have been found to increase markedly the rate of decarboxylation of several jS-keto acids, but to have no effect on the decarboxylation of ketomonocarboxylic acids such as acetoacetic acid. Moreover, only those fl-keto acids having a second carboxylic acid group adjacent to the /3-keto group are affected by the presence of metal ions, e.g., oxaloacetic or oxalosuccinic acids (90, 116, 166, 170, 190, 191, 208). 

This is an enzyme of the citric acid cycle which should not he confused with isocitrate dehydrogenase E.C. 1.1.1.42, from which it can be distinguished by the latter s requirement for NADP and ability to decarboxylate oxalosuccinate. It may be assayed by u.v. spectroscopy [457] or colorimetrically [458—459]. 

The third step of the citric acid cycle involves the protonation of one of the carboxylate groups of oxalosuccinate, a /3-ketoacid, followed by decarboxylation to form a-ketoglutarate ... 

Note that only one of the three carboxyl groups in oxalosuccinate is beta to the ketone carbonyl it is this carboxyl group that undergoes the decarboxylation. 

The anion (oxalosuccinate) is an intermediate in the isocitrate dehydrogenase reaction of the Tricarboxylic add cycle (see). Isodtrate dehydrogenase catalyses both the oxidation of isodtrate to oxalosuccinate, and its decarboxylation to 2-oxoglutarate. 

The metabolic map (see Fig. 1-13) illustrates the reactions involved in the citric acid cycle. After the condensation of acetyl-CoA with oxaloacetate to form citric acid, this carbon compound is transformed into isocitrate, which is itself oxidized and decarboxylated by the same enzyme to yield a-ketoglutarate and CO2. Oxalosuccinate is an intermediate in this reaction. 

As pyruvic acid decarboxylation constitutes the link between glycolysis and the Krebs cycle, a-ketoglutaric decarboxylation divides the reactions involving 6-carbon acids (citrate, isocitrate, and oxalosuccinate) and those involving 4-carbon acids (succinate, fumarate, and malate). The analogy between the two reactions is not restricted to their role in intermediate metabolism, but extends also to the mechanism of action of the two multiple-enzyme systems. In a-ketoglutaric decarboxylation, the overall reaction leads to the formation of CO2 and succinate. CoA, NAD, thiamine, lipoic acid, and magnesium are requirements for this multiple-enzyme system activity. 

Isocitrate (eryt/iro-(2/ ,35)-isocitrate) is oxidized (isocitrate dehydrogenase, EC 1.1.1.41) to oxalosuccinate and the latter undergoes decarboxylation to a-... 

It now appears that both the oxidation and decarboxylation occur simultaneously, that is, without the appearance of an intermediate that dissociates from the enzyme. The mechanism by which these processes occur is not apparent. A possible intermediate in the over-all reaction is oxalosuccinate (VI). This compound has been synthesized and found to... 

The decarboxylation of the j3-keto acids oxalosuccinate and oxal-acetate catalyzed either by appropriate enzymes or by polyvalent cations can be measured spectrophotometrically by the appearance of absorption bands at higher wavelengths than those characteristic of the substrates alone. i These transient bands were once thought to represent complexes of metal with the enol form of the substrate. Studies with model compounds by Steinberger and Westheimer showed, however, that a compound that cannot form an enol, j3, 3-dimethyloxalacetate, is also decarboxylated by metals to form a transient product that has an increased ultraviolet absorption. They identified the absorbing material with the enol of the product. The mechanism of 8-keto acid decarboxylation is therefore as shown in (XI). 

Isocitric dehydrogenase (TPN). 6. Oxalosuccinic decarboxylase (Mn++). 7. Oxidative decarboxylation (CoA, DPN, DPT, thioctic acid). 8. Formation of a phosphorylated derivative of CoA (HjPOJ. 9. Succinic dehydrogenase. 10. Fumarase. 

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