Carboxylation of phosphoenol-pyruvate

Some subaerial plants use a different pathway, involving carboxylation of phosphoenol pyruvic acid (PEP) instead of ribulose diphosphate (the C02 is actually transformed into bicarbonate before incorporation), which subsequently forms a C4 compound, oxaloacetic acid, instead of PGA (Fig. 1.7). Consequently such plants are termed C4 plants. The C4 path is a relatively recent evolutionary development of particular advantage in hot dry climates (see Box 1.10).The PEP cycle effectively transfers C02 to the Calvin cycle, and each cycle confers an isotopic fractionation. Some plants, the CAM plants (see Box 1.10), can use the combined PEP-Calvin cycle path (with some leakage of C02 out of the cell between the cycles) or just the Calvin cycle. The effects of these pathways on the overall isotopic fraction are reflected in the 813C values in Table 5.8. 

PEPC catalyses carboxylation of phosphoenol pyruvate (PEP), employing bicarbonate as carboxyl donor (Cooper and Wood, 1971). As in all such enzymes employing bicarbonate in place of CO2, the enzyme must activate bicarbonate towards carboxyl transfer. In this respect, as in others, PEPC resembles biotin-dependent carboxylases. Two distinct mechanisms are postulated for PEPC one involving the intermediacy of carboxyphosphate, the other a cyclic transition state and pseudorotation at phosphorus. 

Cooper, T.G., Tchen,T.T., Wood,H.G., Benedict, C.R. The carboxylation of phosphoenol-pyruvate and pyruvate. I. The active species of CO2 utilized by phosphoenolpyruvate carboxykinase, carboxytransphosphorylase, and pyruvate carboxylase. J. Biol. Chem. 243,3857-3863 (1968)... 

Oxaloacetic acid, the starter compound of both the TCA and the glyoxylate cycles is regenerated from malic acid under the action of malate dehydrogenase. In green plants, oxaloacetic acid can normally be supplied in any amount needed for operation of the cycles from phosphoenol pyruvate. Oxaloacetic acid is the initial product of CO2 fixation in C4-photosynthetic and Crassulacean acid pathways. In these pathways, oxaloacetic acid is formed by yff-carboxylation of phosphoenol pyruvate catalyzed by phosphoenol pyruvate carboxylase. 

Scheme 9.6 Carboxylation of phosphoenol pyruvate. Adapted from [11]. Copyright 2009 RSC...

The phosphoenol pyruvate so formed is carboxylated by phosphoenol pyruvate oxaloacetate lyase and oxaloacetate is obtained. The latter is transformed into 2 equiv. of acetyl-CoA, 1 equiv. of which re-enters the cycle. 

Today the metabolic network of the central metabolism of C. glutamicum involving glycolysis, pentose phosphate pathway (PPP), TCA cycle as well as anaplerotic and gluconeogenetic reactions is well known (Fig. 1). Different enzymes are involved in the interconversion of carbon between TCA cycle (malate/oxaloacetate) and glycolysis (pyruvate/phosphoenolpyruvate). For anaplerotic replenishment of the TCA cycle, C. glutamicum exhibits pyruvate carboxylase [20] and phosphoenol-pyruvate (PEP) carboxylase as carboxylating enzymes. Malic enzyme [21] and PEP carboxykinase [22,23] catalyze decarboxylation reactions from the TCA cycle... 

Many other carboxylation reactions exist (Barton et al., 1991). For example, in methylo-trophic bacteria, formaldehyde and CO2 are combined to produce acetyl-CoA in the serine or hydroxypyruvate pathway. In contrast, the ribulose monophosphate cycle, which is another methylotrophic pathway of formaldehyde fixation, does not involve carboxylation steps. In addition to those described above, commonly found carboxylation reactions include those of pyruvate or phosphoenol pyruvate. In view of several relatively recent discoveries of novel CO2 assimilation pathways (e.g., the hydoxypro-pionate cycle and anaerobic ammonium oxidation) and growing interest in deep-subsurface microbiology, novel pathways of CO2 incorporation may be discovered in the near future. 

A third fate of pyruvate is its carboxylation to oxaloacetate inside mitochondria, the first step in gluconeogenesis. This reaction and the subsequent conversion of oxaloacetate into phosphoenol pyruvate bypass an irreversible step of glycolysis and hence enable glucose to he synthesized from pyruvate. The carboxylation of pyruvate is also important for replenishing intermediates of the citric acid cycle. Acetyl CoA activates pyruvate carboxylase, enhancing the synthesis of oxaloacetate, when the citric acid cycle is slowed by a paucity of this intermediate. A fourth fate of pyruvate is its oxidative decarboxylation to acetyl CoA, as described on page 763. 

According to Halestrap [98] activation of mitochondrial electron transport not only increases the proton-motive force but also the intramitochondrial ATP concentration which is important for intramitochondrial ATP utilising reactions like pyruvate carboxylation and citrulline synthesis, processes known to be activated by glucagon. Siess et al. [35], however, showed that in hepatocytes glucagon not only increased mitochondrial ATP, but also the sum of ATP, ADP and AMP at the expense of the cytosolic pool of adenine nucleotides, a phenomenon to which no attention has been paid in the literature. Exchange between ADP and ATP cannot increase the mitochondrial adenine nucleotide pool. Possibly net influx of adenine nucleotides can occur via exchange between ADP and mitochondrial phosphoenol-pyruvate (see [4] for literature). 

The key reaction was recognized by Utter to be the phosphorylation of oxaloacetate. Formation of the enol is facilitated in this compound and phosphoenol-pyruvate is then formed by phosphorylation with inosine triphosphate (ITP) (decarboxylation occurs at the same time). Oxaloacetate itself is provided by the direct carboxylation discussed above via a biotin enzyme. Alternately, the reductive carboxylation yields malate which is converted to oxaloacetate. The first reaction, although requiring additional ATP, appears to be the more important one, and, of course, the equilibrium favors C02-fixation more strongly in this way. The following... 

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