Oxalacetate 0-carboxylase

Utilizing the C 0 incorporation in oxalacetate as the enzyme assay. Utter and Kurahashi (357) obtained piu-ified preparations of oxalacetic carboxylase from chicken liver free of malic enzyme and... 

Enzymes, apparently unrelated to the malic enzyme or to Utter s oxalacetic carboxylase, catalyzing the decarbmqrlation of oxalacetate, are present in bacteria and certain plant tissues. Some of these enzymes, including the highly purified oxalacetic carboxylase (decarboxylase) of Microeoceus lysodeikticm (152), can catalyze an incorporation of CO in oxalacetate which does not require ATP (236) the mechanism of this incorporation is unknown. Kalten-bach and Kalnitsky (198,199) have reported a net synthesis of oxal-... 

In 1941, Krampitz and Werkman separated an enzyme preparation (oxalacetate /3-carboxylase) from Micrococcus lysodeikiicus which decar-boxylates oxalacetic acid to form pyruvic acid. Cocarboxylase was not required for this reaction. When oxalacetate, the enzyme and magnesium ions were incubated in the presence of until approximately half of the oxalacetate disappeared, was found in the remaining oxalacetate. The isotope was found exclusively in the carboxyl group adjacent to the methylene group. Since the reaction medium contained only oxalacetate and CO2, and since the only products formed were pyruvic acid and COs, it was concluded that this reaction was reversible. 

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. 

Production of oxalacetate by C02 addition to pyruvic acid, operated by pyruvate carboxylase (biotin coenzyme), allows the cycle to continue. 

Figure 2. Effect of inorganic Mn and pyruvate carboxylase Mn on the proton NMR spectra of (A) pyruvate (Ref. 15) and (B) oxalacetate (Ref. 19)...

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)...

Pyruvate carboxylase-Mn-pyruvate Pyruvate carboxylase-Mn-oxalacetate Pyruvate carboxylase-Mn-a ketobutyrate... 

The latter may take place in the mitochondria and the phosphoenolpyruvate then passes into the cell cytoplasm. Alternatively, oxalacetate may be transferred into the cell cytoplasm by means of the glutamate-aspartate shuttle and converted to phosphoenolpyruvate by cytoplasmic phosphoenolpyruvate carboxykinase. In the ruminant animal, pyruvate carboxylase is located in the cell cytoplasm as well as the mitochondria, and pyruvate can be changed to phosphoenolpyruvate entirely in the cytoplasm. 

Phosphopyruvaie Carboxylases. In addition to the malic enzyme, other oxalacetic decarboxylases are known. Enzymes purified from various... 

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. 

Malate Inhibition (see Table 4.3). Following our initial observation that L-malate and oxalacetate, and to a lesser extent D-malate, aspartate, and glutamate inhibit maize root PEP carboxylase (see Ting, 1971), Queiroz (1967) reported that the PEP carboxylase of Kalanchoe leaves was sensitive to L-malate. Based on these data, it was proposed that L-malate could regulate its own synthesis by feedback inhibition of PEP carboxylase (Queiroz, 1967). It is possible that malate is simply a product analog, acting similarly to oxalacetate, yet in maize roots, oxalacetate and malate inhibit in a different manner (Ting, 1968 a). 

Strangely, we had never turned our attention to determining how CO2 is fixed by the propionic acid bacteria and in 1961 when we did get to this problem we discovered, to our surprise, a new enzyme for fixation of CO2. There were three enzymes which were known to fix CO2 into oxalacetate at that time, P-enolpyruvate carboxylase discovered by R. S. Bandurski and G. M. Griener in 1953,... 

Abbreviations NTP nucleotide triphosphate, PCK phosphoenolpyruvate carboxykinase, NDP nucleotide diphosphate, PEP phosphoenolpyruvate, PPC phosphoenolpyruvate carboxylase, OAA oxalacetic acid or oxalacetate and PYC pyruvate carboxylase... 

Figures 4 and 5 illustrates the use of NMR spectroscopy to study the metabolism of (l-i C)glu-cose in primary cultures of neurons and astrocytes. A simplified scheme of the metabolism of (l-i C)glu-cose in neural cells is given in Figure 4. Briefly, (1-i C)glucose is metabolized to (3- C)pyruvate through the Embden Meyerhoff glycolytic pathway. The (3-i C)pyruvate produced can be transaminated to (3- C)alanine, reduced to (3-i C)lactate or enter the tricarboxylic acid (TCA) cycle through the pyruvate dehydrogenase (PDH) or pyruvate carboxylase (PC) activities. A net increase in (3-i C)lactate reveals increased aerobic glycolysis and is normally observed under hypoxic conditions in normal cells. If (3-i C)pyruvate enters the TCA cycle though PDH it produces (2-i C)acetyl-coenzyme A first, and subsequently (4-i C)a-ketoglutarate. In contrast, if (3-i C)pyruvate is carboxylated to (3-i C)oxalacetate by...

The reaction is catalysed by pyruvate carboxylase, an enzyme requiring biotin and found in liver, kidney and, in smaller amounts, brain and adipose tissue [134]. Oxalacetic acid enters... 

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