Oxalacetate formation from

This is one of three enzymes originally considered to be a single malic enzyme responsible for the conversion of malate to pyruvate. This enzyme also decarboxylates added oxalacetate. The other two enzymes utilise NAD instead of NADP. One (E.C. 1.1.1.38) also decarboxylates oxalacetate the other (E.C. 1.1.1.39) does not. Radiochemical assay may be accomplished by measuring malate formation from pyruvate and C labelled bicarbonate [496]. 

Hunter FE, Leloir LF (1945) Citric acid formation from acetoacetic and oxalacetic acids. J Biol Chem 159 295-310... 

Figure 3 Gradient separation of anions using suppressed conductivity detection. Column 0.4 x 15 cm AS5A, 5 p latex-coated resin (Dionex). Eluent 750 pM NaOH, 0-5 min., then to 85 mM NaOH in 30 min. Flow 1 ml/min. 1 fluoride, 2 a-hydrox-ybutyrate, 3 acetate, 4 glycolate, 5 butyrate, 6 gluconate, 7 a-hydroxyvalerate, 8 formate, 9 valerate, 10 pyruvate, 11 monochloroacetate, 12 bromate, 13 chloride, 14 galacturonate, 15 nitrite, 16 glucuronate, 17 dichloroacetate, 18 trifluoroacetate, 19 phosphite, 20 selenite, 21 bromide, 22 nitrate, 23 sulfate, 24 oxalate, 25 selenate, 26 a-ketoglutarate, 27 fumarate, 28 phthalate, 29 oxalacetate, 30 phosphate, 31 arsenate, 32 chromate, 33 citrate, 34 isocitrate, 35 ds-aconitate, 36 trans-aconitate. (Reproduced with permission of Elsevier Science from Rocklin, R. D., Pohl, C. A., and Schibler, J. A., /. Chromatogr., 411, 107, 1987.)...

There are also voices critical of the rTCA cycle Davis S. Ross has studied kinetic and thermodynamic data and concludes that the reductive, enzyme-free Krebs cycle (in this case the sequence acetate-pyruvate-oxalacetate-malate) was not suitable as an important, basic reaction in the life evolution process. Data on the Pt-catalysed reduction of carbonyl groups by phosphinate show that the rate of the reaction from pyruvate to malate is much too low to be of importance for the rTCA cycle. In addition, the energy barrier for the formation of pyruvate from acetate is much too high (Ross, 2007). 

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... 

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. 

Since in the citric acid cycle there is no net production of its intermediates, mechanisms must be available for their continual production. In the absence of a supply of oxalacetic acid, acctaic" cannot enter the cycle. Intermediates for the cycle can arise from the carinxylation of pyruvic acid with CO, (e.g., to form malic acid), the addition of CO > to phosphcnnlpyruvic acid to yield oxalacetic acid, the formation of succinic acid from propionic acid plus CO, and the conversion of glutamic acid and aspartic acid to alpha-ketoglutaric acid and oxalacetic acid, respectively. See Fig. 3. 

Aldol reactions of this type, involving 2-acetamido-2-deoxyaldohexoses, have been studied in connection with the chemical synthesis of A -acetyl-neuraminic acid (50) and related substances, and, for this reason, the choice of the dicarbonyl compound has thus far been limited to oxalacetic acid and its esters. Oxalacetic acid condenses readily with 2-acetamido-2-deoxyaldohexoses in aqueous solution at pH 11. Under these conditions, acetamido sugars partially epimerize, and the aldol reaction takes place for both of the 2-acetamido-2-deoxyaldohexoses present. The complexity of the reaction is further increased by the formation of asymmetric centers at carbon atoms 3 and 4 of the condensation products, namely, diacids (45) and (48), and this can result in the formation of four diastereo-isomers from each sugar. The reaction using 2-acetamido-2-deoxy-o-rnannose (47) has been the one most extensively studied. In this... 

Figure 3. Energy diagrams for the formation of the pyruvate carboxylase-Mn-pyruvate and -oxalacetate bridge complexes based on the mechanism of Equation 8 (from Ref. 19)...

The coupling constant is inconsistent with carboxyl coordination but consistent with carbonyl coordination 15). Similar data for -ketobu-tyrate 15) and oxalacetate (19) have been fit by exchange contributions (1/tm) and inner sphere contributions Tm and T2m)- The rates of formation of these metal bridge complexes from an outer sphere complex ( 3,4) are limited predominantly by the rate of dissociation of a water molecule from the coordination sphere of the enzyme-bound manganese (Figure 3, Table V) (15,19), as required by the Sj l-outer sphere mechanism of Eigen and Tamm (20),... 

Aminooxyacetate, an inhibitor of glutamate— oxalacetate transaminase, inhibits the formation of aspartate. Soling Kleinicke (1976) observed that aminooxyacetate did not inhibit the formation of glucose from lactate and, therefore, concluded that the malate-aspartate shuttle was not essential for the lactate gluconeogenesis in avian liver. However, Ochs Harris (1980) found that aminooxyacetate did block lactate gluconeogenesis when lower concentrations of pyruvate were used and incubation was for longer than 15 min. They concluded that the malate-aspartate shuttle was required. 

Polyhydroxyalkanoates biosynthesis is regulated, on one hand, by the activity of 3-ketothiolase (EC 2.3.1.16), and on the other hand of acetoacetyl-CoA reductase (EC 1.1.1.36) intracellular PHA breakdown is dependent on the activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30). Besides these three enzymes, the following compounds can be pointed out as major factors responsible of the activities of the key enzymes acetyl-CoA, free CoA, NAD(P) + (or NAD(P)H2, respectively) and, to a lower extent, ATP, pyruvate and oxalacetate. In any case, acetyl-CoA can be considered as the central metabolite both for biomass formation and PHB biosynthesis. This compound stems from the catabolic break down of carbon substrates like sugars (mainly catabolized by the 2-Keto-3-desoxy-6-phosphogluconate pathway) or fatty acids (converted by 6-oxidation). 

The transferases are a large group of enzymes that catalyse the transfer of groups such as acetyl, amino and phosphate from one molecule to another. For example, in the formation of citrate from oxalacetate during the release of energy in the body, addition of an acetyl group takes place in the presence of citrate synthetase ... 

The former is called malic dehydrogenase. Although it was reported early that the enzyme is specific for DPN, in fact malic dehydrogenase from several sources has been found to react with TPN at 5-7 per cent of the rate of the DPN reaction. The affinities for DPN and TPN are quite similar, Kn equals about 10 at neutral pH. The equilibrium of the reaction at neutral pH values lies far to the side of malate and DPN, but, as discussed previously, at higher pH values the equilibrium of reactions in which H+ participates is shifted, and near pH 10 the oxidation of malate proceeds to a considerable extent and at a good rate. Even at low pH values, however, the oxidation of malate is readily carried out when coupled with an effective system for oxidizing DPNH or removing oxalacetate (as citrate formation). 

Krebs Cycle and Fatty Acid Oxidation. A possible role of Krebs cycle intermediates in supporting fatty acid oxidation is now apparent. Complete oxidation to CO2 requires oxalacetate to introduce acetyl CoA into the citric acid cycle. But even the formation of acetoacetate requires the continued generation of ATP to support the activation of fatty acids. The transfer of electrons from fatty acid to oxygen is coupled with phosphate esterification, so that fatty acid oxidation has the theoretical capacity to be self-supporting. In the crude systems that contain all of the essential factors for fatty acid oxidation, fatty acid activation must compete with other reactions for the available ATP, and maximum rates of oxidation occur only when additional ATP is generated through operation of the Krebs cycle. 

Since the orotic acid required by Lactobacillus could be replaced partially by carbamylaspartic acid, the latter compound was investigated further as a possible pyrimidine precursor labeled carbamylaspartic acid was as effective as orotic acid in labeling the nucleic acid pyrimidines of this microorganism (339). If ureidosuccinic acid were formed from oxalacetate or aspartate, it would be possible to outline the formation of the pyrimi-... 

First, all the individual stages which constitute the cycle have been demonstrated to occur in muscle tissue, and the rates at which the individual reactions can proceed are sufficient to account for the maximum rate of respiration. The occurrence of some of the reactions in muscle tissue, as already mentioned, has been known since 1911, when Batelli and Stern demonstrated the rapid oxidation of citrate, succinate, fuma-rate, and malate in frog muscle. In 1936 the work of Martiusand Knoop - revealed the mechanism of the conversion of citrate into succinate, and the last major step of the cycle was discovered in 1937, when the formation of citrate from oxalacetate and pyruvate was demonstrated. It was this reaction which made a series of reactions into a cyclic sequence and which linked the series of reactions leading from citrate to oxalacetate with carbohydrate metabolism. 

Wood, Workman, Hemingway, and Nier have pointed out that a minor modification, already contemplated from the start as one of several possibilities, would meet the facts. The main point of the modification is the assumption that the condensation of oxalacetate with pyruvate or a pyruvate derivative yields primarily cfs-aconitate which is directly converted into isocitrate, whereas the formation of citrate is due to a side reaction (Scheme 5). If the rate of the side reaction between citrate and cis-aconitate is slow compared with the rates of the other reactions, it is to be expected that the fixed carbon appears predominantly in the carboxyl group of a-ketoglutaric acid adjacent to the carboxyl group, as is actually the case. 

Studies of the enzymic mechanism of the citric acid synthesis by Stern and Ochoa have directly shown that citric acid, and not aconitic acid, is the primary product. It had earlier been thought that the mechanism of citric acid synthesis might be similar to that of the reaction leading in vitro to the formation of citric acid from oxalacetic and pyruvic acid in the presence of hydrogen peroxide, where oxalocitramalic acid is an intermediate. Martins, however, found this substance to be metabolically inert in animal tissue. Stern and Ochoa found that aqueous extracts of acetone-dried pigeon liver formed citrate when acetate, oxalacetate, ATP, coenzyme A, and Mg or Mn ions were present. Thus the condensation reaction is preceded by the decarboxylation of pyruvic acid and the formation of an active form of acetate. This active acetate, as discussed below, is acetyl coenzyme A. 

In 1945 Lipmann found that a novel coenz3mae—coenzyme A— is required for the enzymic acetylation of sulfanilamide in pigeon liver preparations. Soon afterwards Nachmannsohn and Berman (see also ) found that a coenzyme is also required for the synthe of acetyl choline from choline and acetate in brain tissue, and this was found to be identical with the coenzyme of the acetylation of sulfanilamide, i Subsequently, three other reactions of acetate were found to involve coenzyme A the formation of acetoacetic acid from acetate, the s3mthesis of citrate from oxalacetate and acetate, - and the exchange reaction between acetyl phosphate and inorganic phosphate in bacterial extracts. " Thus, coenzyme A was shown to be a general coenzyme of acetylations, and... 

Stern, Coon, and del Campillo (see also 198a) found evidence for a second mechanism of formation of acetoacetyl CoA, in which succinate participates. In pig heart preparations no citrate is formed from acetoacetate, oxalacetate, CoA, and ATP unless succinate is also present. To explain the effect of succinate the authors suggest that the following reactions take place ... 

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