Oxalacetate decarboxylation

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... 

Dehydrogenase, malate (oxalacetate-decarboxylating) (nicotinamide adenine dinucleotide phosphate) 429b, 922a, 4249... 

After a period of dark CO 2 fixation resulting in the vacuolar accumulation of malic acid and decreased levels of stored carbohydrate, there is a rapid and marked decrease in stored malic acid when the plants experience light (Fig. 3.6). Malate, once mobilized for subsequent metabolic consumption, may inhibit P-enolpyruvate carboxylase (Ting, 1968) and reduce further carboxylation (Kluge, 1969 Queiroz, 1967 Ting and Osmond, 1973 a, b). It is generally assumed that deacidification occurs because of malate decarboxylation (or oxalacetate decarboxylation) and concomitant release of CO2. 

In the experience of the present author, minor deviations from this procedure may result in decreased yields. Oxalacetic acid of high quality is essential, and this should be verified by a melting-point determination prior to use. Decarboxylation of oxalacetate has been reported111 to occur rapidly at pH 7, and it should be kept to a minimum by maintaining the pH as close to 10 as possible when dissolving the oxalacetic acid. A modification of the Comforth reaction is the co-balt(II)-ion-catalyzed condensation of D-erythrose 4-phosphate with oxalacetate to give 3-deoxyheptulosonic acid 7-phosphate112 (as a mixture of the arabino and ribo isomers). Other procedures for the preparation of KDO will be discussed in subsections 3 and 4 of this Section. 

A modified poly(ethylenimine) also acts as an efficient catalyst for decarboxylation (Suh et al., 1976 Spetnagel and Klotz, 1976). In particular, the partially quaternized polymer [SS] catalyzed the decarboxylation of oxalacetic acid in a bifunctional manner (Spetnagel and Klotz, 1976), as shown in (18). The decarboxylation is thought to occur via pre-equilibrium... 

There are two mechanistic possibilities left, either hydride transfer precedes decarboxylation, or vice versa. These two possibilities can be distinguished using Equations 11.51 and 11.53. Within experimental error only Equation 11.51 is consistent with the isotope effect data collected in Table 11.1, thus confirming that the reaction proceeds via a stepwise mechanism with hydride transfer to triphosphate nucleotide (NADP+) and intermediate formation of oxalacetate preceding decarboxylation ... 

C-kinetic isotope effects have been used to determine the degree of C —C breakage in metal and non-metal catalyzed decarboxylation of oxalacetic acid. (Sec. 2.2.2). 

Although the activated benzisoxazole carboxylates serve admirably as substrates to reveal certain mechanistic details of importance in designing a macromolecular catalyst, they are not of biological interest per se. We have also been anxious to achieve catalyses of reactions that occur in a cellular environment. With respect to decarboxylation, we have been focusing on oxalacetate as a suitable substrate. 

In enzymic decarboxylations the mechanistic pathway seems to involve Schiff base formation between an —NH2 from a lysine residue and a C=0 of the keto acid.52 Likewise, with small-molecule primary amines, catalysis of decarboxylation of /3-ketoacids53-58 has been ascribed to a Schiff base intermediate. The overall reaction for oxalacetate is... 

Rates have been monitored by ultraviolet absorption,60 following the decay of oxalacetate, and by enzymatic assays61,62 responding to the production of pyruvate [see (32)]. Figure 19 illustrates the rapid drop in absorbance with time for oxalacetate in the presence of the primary-amine-containing polymer. Even a sixtyfold excess of substrate over primary-amine concentration was completely decarboxylated. This and... 

Fig. 19. Ultraviolet spectrum of oxalacetate during decarboxylation by 6 x 10-2 residue molar PEIQ-NH2. Initial concentration of oxalacetate 5 x 10-4 M.

TABLE XI. Kinetic Parameters for Catalyzed Decarboxylation of Oxalacetate by Modified... 

Fig. 20. Rate-constant-pH profile for PEIQ-NH2 catalyzed decarboxylation of oxalacetate.

The number of nucleophilic sites per macromolecule n can be established from kinetic measurements in the presence of excess substrate and complementary ones in the presence of excess polymer.48 In the decarboxylation of oxalacetate, n is 90 at pH 4.5 and 150 at pH 7.0. The modified polyethylenimine polymer used contains 140 primary amine groups per macromolecule. Thus 65 to 100% of these act as nucleophilic sites for the decarboxylation reaction. 

It became of interest to see if we could obtain any indication of Schiff base formation with the polymer. Since spectroscopic probes would be obscured with the actual substrate, oxalacetate, because of the progress of the decarboxylation reaction (32), we have examined instead the spectra of oxalacetate-4-ethyl ester in solutions of the same modified poly-(ethylenimine) PEIQ—NH2. Such solutions develop a new absorption band at 290 nm. Furthermore, this band is essentially abolished if NaBH4 is added to the solution (Fig. 21). As is well known, NaBH4 reduces Schiff base linkages to amine groups.43-44... 

It has also been possible to confirm the presence of the reduction product of a Schiff base on the polymer by proton magnetic resonance. For this purpose we have used unmodified poly(ethylenimine), since it too catalyzes the decarboxylation of oxalacetate to its product, pyruvate. Unmodified polyethylenimine was mixed with oxalacetate-4-ethyl ester. One-half of this solution was treated with NaBH4 the second half was exposed to a similar environment but no NaBH4 was added. The borohydride-treated polymer exhibited a strong triplet in the nmr spectrum centered at 3.4 ppm upfield from the HOD resonance. This new feature would be expected from the terminal methyl protons of the oxalacetate ester attached to the polymer. Only a very weak triplet was found in the control sample not treated with borohydride. These observations are strong evidence for the formation of Schiff bases with the polymer primary amine groups. Evidently the mechanistic pathway for decarboxylation by the polymer catalyst is similar to that used enzymatically. 

Yes, we have made some pH activity studies. One example is shown in the article (Fig. 20) for the decarboxylation of oxalacetate. Interestingly, the bell shape is similar to that found by others in the catalysis of this reaction by small-molecule amines. 

Figure 4. Effect of metal ions on nonenzymatic decarboxylation of oxalacetic acid (41)...

However, if a further donor group is introduced, a chelate may be formed that does not involve the carboxylate group to be lost. In these cases, the decarboxylation is dramatically enhanced in the presence of metal ions. This is exactly the situation which pertains with oxalacetic acid, which undergoes a facile metal-promoted decarboxylation (Fig. 5-23). The rate of decarboxylation of oxalacetic acid is accelerated some ten thousand times in the presence of copper(n) salts. The metal ion is thought to play a variety of roles, including the stabilisation of the enolate that is produced after loss of carbon dioxide. 

Figure 5-23. The rate of decarboxylation of oxalacetic acid is accelerated many thousands of times in the presence of metal ions.

The procedure reported in Scheme 13.11 describes deracemization of an amino acid involving oxidation with an L-specific enzyme and transamination with a D-amino transferase using D-aspartate 10, which is generated from L-aspartate 11 by aspartate racemase, as the amino donor. The oxidative enzyme is defined as an L-amino acid deaminase, a flavoprotein from Proteus myxofadens [34]. The transamination reaction is shifted towards the product since the oxalacetate 12 formed decarboxylates spontaneously to give pyruvate and carbon dioxide. 

Precursors. Precursors for this reaction are compounds exhibiting keto-enol tau-tomerism. These compounds are usually secondary metabolites derived from the glycolysis cycle of yeast metabolism during fermentation. Pyruvic acid is one of the main precursor compounds involved in this type of reaction. During yeast fermentation it is decarboxylated to acetaldehyde and then reduced to ethanol. Acetone, ace-toin (3-hydroxybutan-2-one), oxalacetic acid, acetoacetic acid and diacetyl, among others, are also secondary metabolites likely to participate in this kind of condensation reaction with anthocyanins. 

Further proof that COj is assimilated by means of the enzyme oxalacetate /3-carboxylase was obtained by Evans and coworkers, who succeeded in preparing a cell-free preparation of this enzyme from liver. The enz3rme was able to catalyze the decarboxylation of oxalacetate to pyruvate. These investigators were able to demonstrate an uptake of C Oj. Utter and Wood have demonstrated conclusively, however, that in the presence of isotopic CO2, pyruvate can be converted to oxalacetate containing isotopic carbon and that the process is, of course, reversible. Addition of adenosine triphosphate to this liver enzyme system increased the rate of incorporation of C Os. Wood, Vennesland and Evans S have also shown that during the fixation of C02, isotopic carbon is incorporated solely and in equal concentrations into the carboxyl groups of pyruvate, lactate, malate and fumarate. 

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. 

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. 

Wood has recently proposed a condensation of oxalacetate with a two-carbon intermediate as the initial reaction. This proposal, which has also been suggested by Krebs, and Martius, states that pyruvate may be oxidatively decarboxylated to a two-carbon intermediate (not necessarily acetate) which can then condense with oxalacetate to form cfs-aconitate (Fig. 5). Although acetyl phosphate along with other possible two-carbon intermediates has been suggested, no convincing evidence for its participation has as yet been obtained. It is of interest, however, that Buchanan and coworkers have demonstrated by means of C that kidney homogenate oxidizes isotopic acetate and acetoacetate... 

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