PEP carboxylase

In a sort of reciprocal arrangement, the cell also feeds many intermediates back into the TCA cycle from other reactions. Since such reactions replenish the TCA cycle intermediates, Hans Kornberg proposed that they be called anaplerotie reactions (literally, the filling up reactions). Thus, PEP carboxylase and pyruvate carboxylase synthesize oxaloacetate from pyruvate (Figure 20.24). 

FIGURE 20.24 Phosphoenolpyruvate (PEP) carboxylase, pyrnvate carboxylase, and malic enzyme catalyze anaplerotlc reactions, replenishing TCA cycle Intermediates. 

PEP carboxylase occurs in yeast, bacteria, and higher plants, but not in animals. The enzyme is specifically inhibited by aspartate, which is produced by transamination of oxaloacetate. Thus, organisms utilizing this enzyme control aspartate production by regulation of PEP carboxylase. Malic enzyme is found in the cytosol or mitochondria of many animal and plant ceils and is an NADPIT-dependent enzyme. 

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). 

FIGURE 22.30 Essential features of the coinpartinenCation and biochemistry of die Hatch-Slack padiway of carbon dioxide uptake in C4 plants. Carbon dioxide is fixed into organic linkage by PEP carboxylase of meso-phyll cells, forming OAA. Eidier malate (die reduced form of OAA) or aspartate (the ami-iiated form) serves as die carrier transpordiig CO9 to the bundle slieadi cells. Within die bundle slieadi cells, CO9 is liberated by decar-boxyladon of malate or aspartate die C-3 product is returned to die mesophyll cell. 

PEP carboxylase mRNA Ostrem et al., 1987 Unidentified mRNAs Ramagopal, 1987... 

CAM is unlikely to be useful for any of the conventional food crops. Nevertheless, further study of those plants in which CAM is inducible could prove useful since isolation of, for example, the PEP carboxylase gene will permit the isolation of its controlling sequences. This will provide us with another set of stress-specific (drought) promoters and enhancers. Furthermore the PEP carboxylase gene has proved amenable to cloning via the cDNA route (Harpster Taylor, 1986). 

Hofner, R., Vazquez-Morena, L., Winter, K., Bohnert, H.J. Schmitt, J.M. (1987). Induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum by high salinity mass increase and de novo synthesis of PEP-carboxylase. Plant Physiology, 83, 915-19. 

Figure 5 Model of phosphorus (P) deficiency-induced physiological changes associated with the release of P-mobilizing root exudates in cluster roots of white lupin. Solid lines indicate stimulation and dotted lines inhibition of biochemical reaction sequences or mclaholic pathways in response to P deliciency. For a detailed description see Sec. 4.1. Abbreviations SS = sucrose synthase FK = fructokinase PGM = phosphoglueomutase PEP = phosphoenol pyruvate PE PC = PEP-carboxylase MDH = malate dehydrogenase ME = malic enzyme CS = citrate synthase PDC = pyruvate decarboxylase ALDH — alcohol dehydrogenase E-4-P = erythrosc-4-phosphate DAMP = dihydraxyaceConephos-phate APase = acid phosphatase.

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. 

Hydroxycyclopropanecarboxylic acid phosphate HCP 34 is an analogue of phosphoenolpyruvate (PEP) 35 which is metabolized by various enzymes. HCP 34 is a potent competitive inhibitor of enzymes utilizing PEP 35, such as PEP carboxylase, enolase, pyruvate kinase, and probably other enzymes. It is a substantially better inhibitor than phospholactate 36 or phosphoglycolate 37, presumably because of the similarity of its geometric and electronic structures with phosphoenol pyruvate,Eq. 12 [28]. 

The other anaplerotic reactions shown in Table 16-2 are also regulated to keep the level of intermediates high enough to support the activity of the citric acid cycle. Phosphoenolpyruvate (PEP) carboxylase, for example, is activated by the glycolytic intermediate fructose 1,6-bisphosphate, which accumulates when the citric acid cycle operates too slowly to process the pyruvate generated by glycolysis. 

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... 

The PEP carboxylase of mesophyll cells has a high affinity for HCCU (which is favored relative to C02 in aqueous solution and can fix C02 more efficiently than can rubisco). Unlike rubisco, it does not use 02 as an alternative substrate, so there is no competition between C02 and 02. The PEP carboxylase reaction, then, serves to fix and concentrate C02 in the form of malate. Release of C02 from malate in the bundle-sheath cells yields a sufficiently high local concentration of C02 for rubisco to function near its maximal rate, and for suppression of the enzyme s oxygenase activity. 

Three enzymes of the C4 pathway are regulated by light, becoming more active in daylight. Malate dehydrogenase is activated by the thioredoxin-dependent reduction mechanism shown in Figure 20-19 PEP carboxylase is activated by phosphorylation of a Ser residue and pyruvate phosphate dikinase is activated by dephosphorylation. In the latter two cases, the details of how light effects phosphorylation or dephosphorylation are not known. 

In this plant, where would you expect to find (a) PEP carboxylase, (b) rubisco, and (c) starch granules Explain your answers with a model for C02 fixation in these C4 cells. 

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. 

Either of the foregoing mechanisms requires that the ureido anion of biotin attack the rather unreactive carbon atom of carboxyphosphate. Another alternative, which is analogous to that suggested for PEP carboxylase (Eq. 13-53) is for carboxyphosphate to eliminate inorganic phosphate to give the more electrophilic C02 (Eq. 14-9, step a). The very basic inorganic phosphate trianion P043 that is eliminated could remove the proton from Nl of biotin to create the biotin ureido anion (step b), which could then add to C02 (step c).22... 

In bacteria and green plants PEP carboxylase (Eq. 13-53), a highly regulated enzyme, is responsible for synthesizing oxaloacetate. In animal tissues pyruvate carboxylase (Eq. 14-3) plays the same role. The latter enzyme is almost inactive in the absence of the allosteric effector acetyl-CoA. For this reason, it went undetected for many years. In the presence of high concentrations of acetyl-CoA the enzyme is fully activated and provides for synthesis of a high enough concentration of oxaloacetate to permit the cycle to function. Even so, the oxaloacetate concentration in mitochondria is low, only 0.1 to 0.4 x 10-6 M (10-40 molecules per mitochondrion), and is relatively constant.65 79... 

Reductive citric acid cycle 5 3 NAD(P)H, 1 unknown donor", 2 ferredoxin 2-Oxoglutarate synthase Isocitrate dehydrogenase6 Pyruvate synthase PEP carboxylase C02 C02 C02 HCOJ Acetyl-CoA, pyruvate, PEP, oxaloacetate, succinyl-CoA, 2-oxoglutarate 2-Oxoglutarate synthase, ATP-citrate lyase... 

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). 

Singh, R. (1989). Carbon dioxide fixation by PEP carboxylase in pod-walls of chickpea. In Photosynthesis, Molecular Biology and Bioenergetics, ed. G.S. Singhal et al., pp. 315-29. New Delhi Narosa Publishing House. 

Winter, K. (1982). Regulation of PEP Carboxylase in CAM plants. In Crassulacean Acid Metabolism, ed. I.P. Ting M. Gibbs, pp. 153-69. Rockville, MD American Society of Plant Physiologists. 

PEP carboxylase has a lower Km for C02 than does Rubisco. Further, 02 is a very poor substrate for this enzyme. This means that, at relatively low concentrations of C02, the delivery of carbon into photosynthesis products is more efficient than in C3 plants and oxygenation doesn t occur. 

PEP carboxylase is concentrated in special mesophyll cells in the outer part of the leaf. This means that the cells most exposed to the atmosphere are the most efficient at converting C02 into organic products. Photosynthesis involving Rubisco is more prominent in the bundle sheath cells located in the inner part of the leaf around the veins that carry compounds between different parts of the plant. 

After PEP carboxylase makes the oxaloacetate, it is transported to the bundle sheath cells. First, NADPH reduces it to malate, and it is then transported to the bundle sheath cells. In the bundle sheath cells, malic enzyme cleaves the malate to pyruvate and C02 for Rubisco. This generates NADPH as well, so the C4 cycle consumes no reducing equivalents. Pyruvate is transported from the bundle sheath back to the mesophyll cells where it is rephosphorylated to phosphoenolpyruvate, expending the equivalent of two ATP high-energy phosphates. ... 

---