Gluconeogenesis:- Conversion of Pyruvate to Phosphoenolpyruvate Requires Two Exergonic Reactions
The first of the bypass reactions in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvate (PEP). This reaction cannot occur by reversal of the pyruvate kinase reaction of glycolysis (p. 532), which has a large, negative standard free-energy change and is irreversible under the conditions prevailing in intact cells (Table 14–2, step 10). Instead, the phosphorylation of pyruvate is achieved by a roundabout sequence of reactions that in eukaryotes requires enzymes in both the cytosol and mitochondria. As we shall see, the path way shown in Figure 14–16 and described in detail here is one of two routes from pyruvate to PEP; it is the pre dominant path when pyruvate or alanine is the glucogenic precursor. A second pathway, described later, pre dominates when lactate is the glucogenic precursor. Pyruvate is first transported from the cytosol into mitochondria or is generated from alanine within mitochondria by transamination, in which the -amino group is removed from alanine (leaving pyruvate) and added to an -keto carboxylic acid (transamination reactions are discussed in detail in Chapter 18). Then pyruvate carboxylase, a mitochondrial enzyme that requires the coenzyme biotin, converts the pyruvate to oxaloacetate (Fig. 14–17):
Pyruvate+HCO-3+ATP→oxaloacetat + ADP + Pi (14–4)
The reaction involves biotin as a carrier of activated HCO-3(Fig. 14–18). The reaction mechanism is shown in Figure 16–16. Pyruvate carboxylase is the first regulatory enzyme in the gluconeogenic pathway, requiring acetyl-CoA as a positive effector. (Acetyl-CoA is produced by fatty acid oxidation, and its ac cumulation signals the availability of fatty acids as fuel.) As we shall see in Chapter 16 (see Fig. 16–15), the pyruvate carboxylase reaction can replenish intermediates in another central metabolic pathway, the citric acid cycle.
Because the mitochondrial membrane has no trans porter for oxaloacetate, before export to the cytosol the oxaloacetate formed from pyruvate must be reduced to malate by mitochondrial malate dehydrogenase, at the expense of NADH:
FIGURE 14–17 Synthesis of phosphoenolpyruvate from pyruvate. (a)In mitochondria, pyruvate is converted to oxaloacetate in a biotin requiring reaction catalyzed by pyruvate carboxylase. (b)In the cytosol, oxaloacetate is converted to phosphoenolpyruvate by PEP carboxy kinase. The CO2 incorporated in the pyruvate carboxylase reaction is lost here as CO2. The decarboxylation leads to a rearrangement of electrons that facilitates attack of the carbonyl oxygen of the pyruvate moiety on the phosphate of GTP.
FIGURE 14–18 Role of biotin in the pyruvate carboxylase reaction. The cofactor biotin is covalently attached to the enzyme through an amide linkage to the -amino group of a Lys residue, forming a biotinyl-enzyme. The reaction occurs in two phases, which occur at two different sites in the enzyme. At catalytic site 1, bicarbonate ion is converted to CO2 at the expense of ATP. Then CO2reacts with biotin, forming carboxybiotinyl-enzyme. The long arm composed of biotin and the side chain of the Lys to which it is attached then carry the CO2 of carboxybiotinyl The standard free-energy change for this reaction is quite high, but under physiological conditions (including a very low concentration of oxaloacetate) G ≈ 0 and the reaction is readily reversible. Mitochondrial malate dehydrogenase functions in both gluconeogenesis and the citric acid cycle, but the overall flow of metabolites in the two processes is in opposite directions. Malate leaves the mitochondrion through a specific transporter in the inner mitochondrial membrane (see Fig. 19–27), and in the cytosol it is reoxidized to oxaloacetate, with the production of cytosolic NADH:-enzyme to catalytic site 2 on the enzyme surface, where CO2 is released and reacts with the pyruvate, forming oxaloacetate and regenerating the biotinyl-enzyme. The general role of flexible arms in carrying reaction intermediates between enzyme active sites is described in Figure 16–17, and the mechanistic details of the pyruvate carboxylase reaction are shown in Figure 16–16. Similar mechanisms occur in other biotin-dependent carboxylation reactions, such as those catalyzed by propionyl-CoA carboxylase (see Fig. 17–11) and acetyl-CoA
Malate+NAD+→ oxaloacetate NADH H (14–6)
The oxaloacetate is then converted to PEP by phosphoenolpyruvate carboxykinase (Fig. 14–17). This Mg2+-dependent reaction requires GTP as the phosphoryl group donor:
The reaction is reversible under intracellular conditions; the formation of one high-energy phosphate compound (PEP) is balanced by the hydrolysis of another (GTP). The overall equation for this set of bypass reactions, the sum of Equations 14–4 through 14–7, is
Two high-energy phosphate equivalents (one from ATP and one from GTP), each yielding about 50 kJ/mol under cellular conditions, must be expended to phosphorylate one molecule of pyruvate to PEP. In contrast, when PEP is converted to pyruvate during glycolysis, only one ATP is generated from ADP. Although the standard free energy change (ΔG0) of the two-step path from pyruvate to PEP is 0.9 kJ/mol, the actual free-energy change (ΔG), calculated from measured cellular concentrations of intermediates, is very strongly negative (-25 kJ/mol); this results from the ready consumption of PEP in other reactions such that its concentration remains relatively low. The reaction is thus effectively irreversible in the cell. Note that the CO2 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). There is a logic to the route of these reactions through the mitochondrion. The [NADH]/[NAD+] ratio in the cytosol is 8x10-4, about 105 times lower than in mitochondria. Because cytosolic NADH is consumed in gluconeogenesis (in the conversion of 1,3-bisphos phoglycerate to glyceraldehyde 3-phosphate; Fig. 14–16), glucose biosynthesis cannot proceed unless NADH is available. The transport of malate from the mitochondrion to the cytosol and its reconversion there to oxaloacetate effectively moves reducing equivalents to the cytosol, where they are scarce. This path from pyruvate to PEP therefore provides an important balance between NADH produced and consumed in the cytosol during gluconeogenesis.
A second pyruvate → PEP bypass predominates when lactate is the glucogenic precursor (Fig. 14–19). This pathway makes use of lactate produced by glycolysis in erythrocytes or anaerobic muscle, for example, and it is particularly important in large vertebrates after vigorous exercise (Box 14–1). The conversion of lactate to pyruvate in the cytosol of hepatocytes yields NADH, and the export of reducing equivalents (as malate) from mitochondria is therefore unnecessary. After the pyruvate produced by the lactate dehydrogenase reaction is transported into the mitochondrion, it is con verted to oxaloacetate by pyruvate carboxylase, as described above. This oxaloacetate, however, is converted directly to PEP by a mitochondrial isozyme of PEP carboxykinase, and the PEP is transported out of the mitochondrion to continue on the gluconeogenic path. The mitochondrial and cytosolic isozymes of PEP carboxy kinase are encoded by separate genes in the nuclear chromosomes, providing another example of two dis tinct enzymes catalyzing the same reaction but having different cellular locations or metabolic roles (recall the isozymes of hexokinase).
FIGURE 14–19 Alternative paths from pyruvate to phospho enolpyruvate. The path that predominates depends on the glucogenic precursor (lactate or pyruvate). The path on the right predominates when lactate is the precursor, because cytosolic NADH is generated in the lactate dehydrogenase reaction and does not have to be shuttled out of the mitochondrion (see text). The relative importance of the two pathways depends on the availability of lactate and the cytosolic requirements for NADH by gluconeogenesis
