The net effect of gluconeogenesis is the reversal of glycolysis. In gluconeogenesis, glucose is synthesized from smaller precursors. From the G and values, you learned in BB 450, you can have an appreciation for the difficulty a cell has in simply reversing a pathway.
The overall change for all of the reactions of glycolysis in the forward reaction is -73.3 kJ/mol. If one had to reverse the reaction by changing the concentration of the products, it would require over a trillion-fold excess of the product of glycolysis (pyruvate) to the reactant of glycolysis (glucose). This kind of ratio is very hard for the cell to generate. By the time it produced enough pyruvate and used its glucose, it would have starved to death for lack of glucose. See also Table 13.1. Remember that the G values for glycolysis in the way it is written are the negative of those for the reverse reaction.
To get around the problem of controlling pathways simply by reversing all of the reactions in them, the cell performs gluconeogenesis using some of the enzymes from glycolysis for which there is little energy barrier and employs different "bypassing" reactions around the enzymatic reactions of glycolysis for which there is a large energy barrier. Surprisingly, gluconeogenesis accomplishes all of this by using seven enzymes from glycolysis (and reversing those reactions) and four other enzymes that catalyze slightly different reactions that the corresponding three enzymes of glycolysis.
The enzymes unique to gluconeogenesis are Pyruvate Carboxylase (PC) and PEP carboxykinase (PEPCK) instead of Pyruvate Kinase (PK) of glycolysis, fructose bisphosphatase (FBPase) instead of Phosphofructokinase (PFK) from glycolysis, and glucose-6-phosphatase (G6Pase) instead of Hexokinase (HK) from glycolysis. Notice how they bypass large G value reactions of glycolysis (Table 13.1). All of the unique gluconeogenesis enzymes have important regulatory controls on them, as we shall see (see also Figure 16.6), because it is important that gluconeogenesis be turned off when glycolysis is turned on, and vice versa.
Why is it important to have glycolysis off when gluconeogenesis is on? Consider what happens if both pathways are operating in a cycle. (See Figure 16.3) One starts with a glucose molecule, breaks it to 2 pyruvates in glycolysis and then resynthesizes glucose from the two pyruvates via gluconeogenesis. If glycolysis and gluconeogenesis are operating simultaneously in the cell, there is thus an overall net energy loss of 2 ATPs and 2 GTPs and no net gain of anything. It is for this reason that the cell must regulate separately when each pathway occurs. To have both pathways going steadily results in continuous loss of energy and no net change in substrate concentrations. This is known as a futile cycle.
It is important to recognize that the metabolic needs of individual cells, and the metabolic needs of a complete organism vary. (See Figure 16.2). The brain has a constant need for glucose that must be met, even when the body is starving. To accommodate these different demands, gluconeogenesis may be occurring in liver cells to supply glucose to brain cells that use it in glycolysis. Thus glycolysis and gluconeogenesis may be going on at different organs of an organism at the same time. See Cori cycle, Figure 16.5.
Pyruvate is not the only starting point for making glucose in the cell. (See Figure 16.4) As seen in the Cori cycle, lactate, a product of anaerobic metabolism, is an excellent precursor. Others include glycerol (modified to form DHAP), propionyl CoA (converted ultimately to PEP), and amino acids such as alanine (converted to pyruvate), aspartate (ultimately converted to PEP), and 16 others.
Control of the important enzymes of glycolysis/gluconeogenesis is accomplished in three ways -
2) by enzyme modification (phosphorylation - see F2,6BP system)),
3) at the level of protein synthesis of the enzyme (hormonal control of synthesis).
This is illustrated in Figure 16.6. Notice that some allosteric effectors, such as G6P, F2,6BP, acetyl-CoA, and AMP, have opposite effects on enzymes in glycolysis and gluconeogenesis, turning one off as it turns the other on. Others, such as Citrate, ATP, and F1,6BP affect only one of the pathways.
Allosteric regulation is organized so that molecules (like ATP and citrate), which are consistent with a "high" energy state of the cell, turn off glycolysis. Acetyl-CoA is consistent with a high energy state, and it turns off glycolysis and turns on gluconeogenesis as well. Conversely, molecules, such as AMP, which are indicative of a "low" energy state of the cell, turn on glycolysis and turn off gluconeogenesis. Coordinated controls insure than within a cell that futile cycles of glycolysis/gluconeogenesis are not occurring.
An enzyme system external to glycolysis and gluconeogenesis synthesizes and degrades a potent allosteric effector of the PFK/FBPase control point. (This system is illustrated in Figure 16.7). The effector is fructose 2,6 bisphosphate (F2,6BP).
F2,6BP is a strong activator of PFK-1 (glycolysis) and an inhibitor of FBPase (gluconeogenesis). F2,6BP is synthesized from F6P by Phosphofructokinase-2 (PFK-2) and degraded to F6P by Fructose bisphosphatase-2 (FBPase-2).
PFK-2 and FBPase themselves are allosterically regulated. The allosteric effectors of PFK-2 and FBPase - citrate (inhibits PFK-2), AMP (activates PFK-2), F6P (inhibits FBPase-2, activates PFK-2), and glyceraldehyde 3 phosphate (activates FBPase-2) - are all directed towards managing the cell's energy needs - inhibiting PFK-2/activating FBPase-2 when cells have plenty of energy and activating PFK-2/inhibiting FBPase-2 when cells need metabolic energy.
In addition to these controls, the activities of PFK-2 and FBPase-2 are regulated by covalent modification of the enzyme via phosphorylation. PFK-2 is inactivated by phosphorylation (high energy state) and FBPase-2 is activated by phosphorylation. Thus, F2,6BP is an activator of glycolysis and an inactivator of gluconeogenesis.
Allosteric Regulators of Glycolysis/Gluconeogenesis control enzymes.
Citrate - produced by citric acid cycle - indicator of high energy - inhibits PFK and PFK-2 (turns off glycolysis).
ATP - produced in glycolysis - indicator of high energy - inhibits PFK (turns off glycolysis).
AMP - produced in making ATP from 2ADPs when cells low in ATP - indicator of low energy - inhibits FBPase, activates PFK and PFK-2 (turns on glycolysis).
Acetyl-CoA - indicator of high energy - activates PC (turns on gluconeogenesis)
F6P - activates PFK-2, inhibits FBPase-2 (turns on glycolysis, turns off gluconeogenesis).
F1,6BP - activates PFK(turns on glycolysis)
Alanine - from breakdown of proteins - inhibits PK (turns off glycolysis)
Oxaloacetate (OAc) is synthesized from pyruvate by pyruvate carboxylase only in the mitochondria, whereas the location of the enzymes that convert PEP to glucose are only present in the cytoplasm. OAc cannot be transferred across the mitochondrial membrane because the membrane is impermeable to it. In order for OAc synthesized in the mitochondria to be used in the cytoplasm, it must be converted into a molecule that can be transported across the mitochondrial membrane, and then converted back to OAc after the transfer. Two mechanisms exist - 1) conversion of OAc to malate via a malate dehydrogenase mechanism, transport, and then conversion of malate to OAc in the cytoplasm, and 2) conversion of OAc to aspartate via an aspartate aminotransferase mechanism, transport, and then conversion of aspartate back to OAc. The differences between the two mechanisms involves the need for NADH. NADH is not required via the aspartate route, but is used in the mitochondria and generated in the cytoplasm via the malate route. NADH is required in the cytoplasm for gluconeogenesis, so a malate route will favor gluconeogenesis. However, if the starting point in gluconeogenesis is lactate instead of pyruvate, abundant NADH is already present from the pyruvate -> lactate reaction, enabling the aspartate mechanism. As we shall see in oxidative phosphorylation, the reversal of the malate process (moving NADH into the mitochondria) is important too.
In addition to regulation of gluconeogenesis by allosterism and protein modification, gluconeogenesis can also be controlled hormonally. As will be seen with glycogen metabolism, two hormones play significant roles. Glucagon stimulates production of cAMP. Remember that cAMP favors degradation of F2,6BP via phosphorylation of PFK-2 to form F2,6BPase. Glucagon also acts to stimulation transcription of the protein PEPCK, an essential enzyme of gluconeogenesis. It also represses synthesis of the glycolysis enzyme, pyruvate kinase. Insulin, on the other hand tends to turn off PEPCK transcription.
1. Reasons for different
controls on gluconeogenesis/glycolysis
2. Recognition of differing needs of cells and the organism (Cori cycle)
3. Understanding coordinated allosteric controls on control enzymes in glycolysis/gluconeogenesis
4. Mechanisms of control of enzyme activities;
5. Mechanism for overcoming the spatial separation of Pyruvate Carboxylase from the other enzymes of gluconeogenesis present in the cytosol.
You should understand why high energy compounds turn on gluconeogenesis and turn off glycolysis. You should also recognize that compounds like ATP and citrate are indicative of high energy in the cell and that AMP and alanine are indicative of low energy in the cell.
External Gluconeogenesis Links:
1. From Italy (http://www.med.unibs.it/~marchesi/gluconeo.html)
2. In 2D (http://www.gwu.edu/~mpb/gluco.htm) and 3D (http://www.gwu.edu/~mpb/gluco3d.htm) Viewing in 3D requires the CHIME plug-in. Views are awesome, but may make your browser crash.
3. Interesting facts (http://mills.edu/RESEARCH/FUTURES/JOHNB/pathways/801.html)
4. Broad view, including Cori cycle (http://web.indstate.edu/thcme/mwking/subjects.html)
5. Molecular models of enzymes of glycolysis and gluconeogenesis. (http://chemistry.gsu.edu/glactone/PDB/Proteins/glycolys/glyco.html)