PEP carboxylation

Mirocha CJ, Schauerhamer B, Pathre SV (1974) Isolation, detection, and quantification of zearalenone in maize and barley. J Assoc Anal Chem 57 1104-1110 Muller R, Baier M, Kaiser WM (1991) Differential stimulation of PEP-carboxylation in guard cells and mesophyll cells by ammonium or fusicoccin. J Exp Bot 42 215-220 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15 473-497... 

CAM employs a biochemical strategy similar to C4 plants in that CO2 is first fixed by carboxylation of PEP to produce malate. The malate is later decarboxylated, and the resulting CO2 is refixed by the RPP cycle. The difference between the CAM and C4 strategies lies in the separation of PEP carboxylation from the RPP cycle. In C4 plants the two processes are separated spatially (mesophyll cells and bundle sheath cells), whereas CAM plants separate the PEP carboxylation from the RPP cycle temporally (night and day). 

The most significant difference between C4 metabolism and CAM is the way in which PEP carboxylation is separated from the Calvin cycle. Recall that in C4 metabolism the two processes are spatially separated (i.e., two cell types are used). In CAM the processes are temporally separated within mesophyll cells. In other words, during daylight hours, CO, is regenerated from malate that was synthesized during the night. 

Other metabolic processes such as PEP-carboxylation in the cytosol appeared to be as insensitive to dehydration as photosynthesis (11). It is therefore difficult to interprete reports pointing to a rather high sensivity to water stress of nitrate reduction or nitrate reductase activity (NRA) in leaves. E.g.,it was shown that a 35% water deficit caused a more than 80% inhibition of NRA in cucumber leaves (5, and literature). Such a water deficit has practically no effect on photosynthesis at high external CO, (compare Fig.l). Therefore it seemed possible that the inhibition of the nitrate reducing system by water stress was a consequence of decreased photosynthesis rates, and not an independent event. This is in fact suggested by the following observations. 

The isomerization of succinyl-CoA to methylmalonyl-CoA is not directly linked to ATP generation. However, the B -dependent enzyme prepares the substrate for decarboxylation that leads to the synthesis of ATP and formation of CO2 and propionate. Through the pivotal action of methylmalonyl-oxaloacetate transcarboxylase the propionic acid bacteria can gain 6 mol ATP from 1.5 mol fermented glucose. Thus, stimulation of methylmalonyl-CoA decarboxylation favors a general increase in energy output. If the decarboxylation cannot proceed, however, the bacteria carry out the PEP-carboxylation reaction, in which one molecule of the macroergic... 

Although the enzymatic machinery for P-enolpyruvate carboxylation in the nonautotrophic pathway, C3-photosynthesis, C4-photosynthesis, and CAM is similar, the enzymes are sufficiently different to be classed as isozymes, and hence, we assume evolutionary pressures have resulted in a common theme (PEP carboxylation) being modified for similar, but functionally different adaptive roles (see Chap. 4). 

Hence, those enzymes involving PEP carboxylation and regeneration, and malate production and degradation plus synthesis of the amino acids aspartate and alanine from the keto acids oxalacetate and pyruvate are deemed most interesting for our understanding in CAM. 

Succinate is an important precursor for plastics, pharmaceuticals, detergents, and other consumer products (da Silva et al, 2009). Several biodegradable plastics can be produced with succinate, and the production of these plastics with renewable resources is desirable. Also, one mole of carbon dioxide is fixed for every mole of succinate produced thus, fermentative production of succinate can be used to reduce carbon dioxide emissions (Song and Lee, 2006). Succinate production can be accomplished using several metabolic pathways, but the most prevalent in bacteria is PEP carboxylation. This is the same pathway previously described for propionate production, with the exception that succinate is not converted to propionate. 

Critical micelle concentration (Section 19 5) Concentration above which substances such as salts of fatty acids aggre gate to form micelles in aqueous solution Crown ether (Section 16 4) A cyclic polyether that via lon-dipole attractive forces forms stable complexes with metal 10ns Such complexes along with their accompany mg anion are soluble in nonpolar solvents C terminus (Section 27 7) The amino acid at the end of a pep tide or protein chain that has its carboxyl group intact—that IS in which the carboxyl group is not part of a peptide bond Cumulated diene (Section 10 5) Diene of the type C=C=C in which a single carbon atom participates in double bonds with two others... 

Compartmentation of these reactions to prevent photorespiration involves the interaction of two cell types, mescrphyll cells and bundle sheath cells. The meso-phyll cells take up COg at the leaf surface, where Og is abundant, and use it to carboxylate phosphoenolpyruvate to yield OAA in a reaction catalyzed by PEP carboxylase (Figure 22.30). This four-carbon dicarboxylic acid is then either reduced to malate by an NADPH-specific malate dehydrogenase or transaminated to give aspartate in the mesophyll cells. The 4-C COg carrier (malate or aspartate) then is transported to the bundle sheath cells, where it is decarboxylated to yield COg and a 3-C product. The COg is then fixed into organic carbon by the Calvin cycle localized within the bundle sheath cells, and the 3-C product is returned to the mesophyll cells, where it is reconverted to PEP in preparation to accept another COg (Figure 22.30). Plants that use the C-4 pathway are termed C4 plants, in contrast to those plants with the conventional pathway of COg uptake (C3 plants). 

C4 plants incorporate CO2 by the carboxylation of phosphoenolpyruvate (PEP) via the enzyme PEP carboxylase to make the molecule oxaloacetate which has 4 carbon atoms (hence C4). The carboxylation product is transported from the outer layer of mesophyll cells to the inner layer of bundle sheath cells, which are able to concentrate CO2, so that most of the CO2 is fixed with relatively little carbon fractionation. 

Note that the C02 added to pyruvate in the pyruvate carboxylase step is the same molecule that is lost in the PEP carboxykinase reaction (Fig. 14-17). This carboxylation-decarboxylation sequence represents a way of activating pyruvate, in that the decarboxylation of oxaloacetate facilitates PEP formation. In Chapter 21 we shall see how a similar carboxylation-decarboxylation sequence is used to activate acetyl-CoA for fatty acid biosynthesis (see Fig. 21-1). 

When intermediates are shunted from the citric acid cycle to other pathways, they are replenished by several anaplerotic reactions, which produce four-carbon intermediates by carboxylation of three-carbon compounds these reactions are catalyzed by pyruvate carboxylase, PEP carboxykinase, PEP carboxylase, and malic enzyme. Enzymes that catalyze carboxylations commonly employ biotin to activate C02 and... 

Amino acids in aqueous solution contain weakly acidic a-carboxyl groups and weakly basic a-amino groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain. Thus, both free amino acids and some amino acids combined in pep tide linkages can act as buffers. The quantitative relationship between the concentration of a weak acid (HA) and its conjugate base (A-) is described by the Henderson-Hasselbalch equation. 

The first "roadblock" to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of pyruvate to phosphoenolpyruvate (PEP) by pyruvate kinase. In gluconeogenesis, pyruvate is first carboxylated by pyruvate carboxylase to oxaloacetate (OAA), which is then converted to PEP by the action of PEP-carboxykinase (Figure 10.3). 

The carboxyl group enters on the si face of PEP. However, there is another possibility.295 296 The carboxyl phosphate, while in the active site adjacent to the enolate anion, may eliminate phosphate, the enolate ion adding to the resulting C02 to form the final product. According to this mechanism the group transfer potential of the phosopho group in PEP is... 

PEP carboxylase is lacking from animal tissues and fungi. In these creatures PEP is converted to pyruvate, which is then carboxylated to oxaloacetate with coupled cleavage of ATP by the action of pyruvate carboxylase (Eq. 14-3), an enzyme that not only utilizes bicarbonate ion but also contains biotin. However, there are mechanistic similarities between its action and that of PEP carboxylase. 

Carboxylation followed by a later decarboxylation is an important pattern in other biosynthetic pathways, too. Sometimes the decarboxylation follows the carboxylation by many steps. For example, pyruvate (or PEP) is converted to uridylic acid (Eq. 17-41 details are shown in Fig. 25-14) ... 

The first step in the conversion of pyruvate to PEP entails the carboxylation of pyruvate to form oxaloacetate. This reaction is catalyzed by pyruvate carboxylase. As in... 

In this malate dismutation pathway, carbohydrates are degraded to phosphoenolpyruvate (PEP) via the classical glycolytic pathway. This PEP is then carboxylated by PEP carboxykinase (PEPCK) to oxaloacetate, which is subsequently reduced to malate. This malate is transported into the mitochondria and is degraded in a split pathway. A portion of the malate is oxidized to acetate and another portion is reduced to succinate, which can then be further metabolized to propionate (Fig. 20.1). 

This cycle resembles the 3-hydroxypropionate/4-hydroxybutyrate cycle, but with pyruvate ferredoxin oxidoreductase (pyruvate synthase) and phosphoenolpyruvate (PEP) carboxylase as the carboxylating enzymes (Figure 3.6). 

The dicarboxylate/4-hydroxybutyrate cycle starts from acetyl-CoA, which is reductively carboxylated to pyruvate. Pyruvate is converted to PEP and then car-boxylated to oxaloacetate. The latter is reduced to succinyl-CoA by the reactions of an incomplete reductive citric acid cycle. Succinyl-CoA is reduced to 4-hydroxybu-tyrate, the subsequent conversion of which into two acetyl-CoA molecules proceeds in the same way as in the 3-hydroxypropionate/4-hydroxybutyrate cycle. The cycle can be divided into part 1 transforming acetyl-CoA, one C02 and one bicarbonate to succinyl-CoA via pyruvate, PEP, and oxaloacetate, and part 2 converting succinyl-CoA via 4-hydroxybutyrate into two molecules of acetyl-CoA. This cycle was shown to function in Igrticoccus hospitalis, an anaerobic autotrophic hyperther-mophilic Archaeum (Desulfurococcales) [40]. Moreover, this pathway functions in Thermoproteus neutrophilus (Thermoproteales), where the reductive citric acid cycle was earlier assumed to operate, but was later disproved (W.H. Ramos-Vera et al., unpublished results). 

Fig. 2 Theoretical influence of pH on the ratio of equilibrium constants FCapp/- pep and Kapp/Kest for peptide (solid line) and ester (dotted line) bond formation, respectively. pKi and pK2 are the acid dissociation constants of the carboxyl and amino groups, respectively, the values used for the calculation (pi i = 3.75, pK2 = 7.75) are representative of an internal peptide bond. Theoretical values calculated using the following equations Kapp/ffpep = 1/(1 + [H+]/fCi)(l + K2/[H+D with Kpep = [peptide]/[RC02-][R NH3+], Kapp/ffest = 1/(1 + Ki/[H+]) with Kest = [ester]/[RC02H][R OH]...

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