Glycolysis & Gluconeogenesis Notes

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Introduction

Glycolysis is a central metabolic pathway involving metabolism of the sugar glucose. THIS shows an overview of the process, being divided into a phase in which ATP energy is invested, a phase in which a six carbon intermediate is broken in to two molecules with three carbons each, and a phase in which ATP energy is generated from the three carbon molecules. The starting point for glycolysis is the molecule glucose and the process ends with formation of two pyruvate molecules. Additional products of glycolysis include two ATPs and two NADHs.

Reactions of Glycolysis

There are ten enzymatic reactions in glycolysis. Students should know the names of the enzymes, whether the for each reaction is large/small, positive/negative, or close to zero. You do not need to know the actual numbers, however. Students also need to know the structure of each non-nucleotide intermediate.

Highlights of the reactions:

Reaction #1

Alpha-D-Glucose + ATP <=> -D-Glucose-6-Phosphate + ADP + H+ / See also HERE

Enzyme: Hexokinase
Notes - ATP energy is used. Hexokinase is capable of phosphorylating other 6-carbon sugars similarly, such as galactose, fructose, and mannose. is negative, so it favors making glucose-6-phosphate (G6P), but the product of the reaction (G6P) can reach high enough concentration to inihibit hexokinase and limit glycolysis. = -16.7 kJ/mol

Reaction #2

-D-glucose-6-phosphate <=> D-fructose-6-phosphate / See also HERE

Enzyme: Phosphoglucoisomerase
This is an aldose-ketose isomerization that proceeds through an enediol intermediate. G6P is the aldose and fructose-6-phosphate (F6) is the ketose. Phosphoglucoisomerase, which catalyzes this isomerization, must not be confused with phosphoglucomutase, the enzyme that interconverts G6P and glucose-1-phosphate (G1P). The for the isomerization of G6P to F6P is only slightly positive, so it strongly favors neither reactants nor products in this reaction.

= +1.7 kJ/mol

Reaction #3

D-fructose-6-phosphate + ATP <=> D-fructose-1,6-bisphosphate / See also HERE

Enzyme: Phosphofructokinase
ATP energy is used to phosphorylate F6P to fructose-1,6-bisphosphate (F1,6BP). This reaction is the key to understanding how regulation of glycolysis is regulated. The enzyme, phosphofructokinase (PFK), is allosterically regulated by AMP (on), ADP (on), ATP (off), citrate (off), and fructose-2,6-bisphosphate (F2,6BP) (on). The most potent of these is F2,6BP. The of -14.2 kJ/mol favors formation of F1,6BP fairly strongly. Consequently, the reaction is essentially irreversible in vivo. At this point all of the energy inputs for glycolysis are complete.

= -14.2 kJ/mol

 

Reaction #4

D-fructose-1,6-bisphosphate <=> Dihydroxyacetone phosphate + D-Glyceraldehyde-3-Phosphate / See also HERE Enzyme: Fructose-1,6-Bisphosphate Aldolase
In this reaction, F1,6BP is cleaved to yield two three-carbon intermediates, glyceraldehyde-3-phosphate (G3P and dihydroxyacetone phosphate (DHAP). The large positive (+23.9 kJ/mol) strongly favors the reverse reaction under conditions where reactants and products are present in relatively equal quantitities. In muscle, however, the concentrations of G3P and DHAP are kept low enough that the forward reaction is favored overall . This is a good example of how a reaction that is unfavorable at standard state conditions can be made favorable in the cell by removing products as they are formed.

: = +23.9 kJ/mol

 

Reaction #5

Dihydroxyacetone phosphate <=> D-Glyceraldehyde-3-Phosphate / See also HERE

Enzyme: Triose Phosphate Isomerase

Notes - The isomerization of DHAP to G3P, like the isomerization of G6P to F6P (reaction 2 above), proceeds through an enediol intermediate. Additionally, the isomerization of DHAP also has a positive , but the reaction is pulled to the right by keeping the cellular concentration of G3P very low.

For all reactions that follow in this section, keep in mind that the six-carbon glucose has been split into two three-carbon units. Thus, to account for everything properly, remember that there are two of each three carbon compound in the reactions shown.

The enzyme catalyzing this reaction is not only one of the most efficient enzymes known, it is also theoretically as fast as an enzyme can get.

= +7.6 kJ/mol

Reaction #6

D-Glyceraldehyde-3-Phosphate + NAD+ + Pi <=> 1,3 bisphosphoglycerate + NADH + H+ / See also HERE Enzyme: Glyceradehyde-3-Phosphate Dehydrogenase
Notes - In this reaction, G3P is phosphorylated and oxidized, so something (NAD+) must be concomitantly reduced. As a result, the NAD+/NADH balance in the cell is important. If the concentration of NAD+ is low, the reverse reaction is favored, preventing glycolysis from occurring aerobically. Instead it must occur anaerobically. Thus this reaction determines whether glycolysis occurs aerobically or anaerobically. 1,3-bisphosphoglycerate (1,3BPG), the reaction product, contains an acylphosphate group, which has a standard free energy of hydrolysis of 49.4kJ/mol. Thus, 1,3BPG is capable of synthesizing ATP via a substrate-level phosphorylation. The slightly positive shows that the reverse reaction is slightly favored at standard conditions. In the cell, however, the forward reaction is favored, thanks partly to the high NAD+/NADH ratio normally present. Note also that the NADH produced in this reaction can be used to make three molecules of ATP in aerobic glycolysis (when oxidative phosphorylation is occurring). Finally, glyceraldehyde-3-phosphate dehydrogenase uses a thiol group in catalysis, which can be inhibited by iodoacetate and heavy metals, such as mercury.

= +6.3 kJ/mol

Reaction #7

1,3 bisphosphoglycerate + ADP <=> 3-phosphoglycerate + ATP / See also HERE

Enzyme: Phosphoglycerate Kinase
Notes - This reaction is a substrate-level phosphorylation of ADP to produce 3-phosphoglycerate (3PG) and the first ATP of glycolysis. Because two molecules of ATP are produced per molecule of glucose, the net yield of ATP is zero at this stage of glycolysis. = -18.8 kJ/mol

 

Reaction #8

3-phosphoglycerate <=> 2-phosphoglycerate / See also HERE

Enzyme: Phosphoglycerate Mutase
 Notes - The for the isomerization slightly favors formation of 3PG over 2PG under standard conditions, but in the cell the concentration of 3PG is kept high relative to the concentration of 2PG, which drives the reaction to the right.

= +4.4 kJ/mol

Reaction #9

2-phosphoglycerate <=> Phosphoenolpyruvate + H2O / See also HERE

Enzyme: Enolase
Notes - This reaction is a simple dehydration (or elimination) of 2PG to form phosphoenolpyruvate (PEP), but it has the effect of increasing the energy of hydrolysis of the phosphate bond almost four fold (from -15.6 kJ/mol in 2PG to -61.9 kJ/mol in PEP). This high free energy of hydrolysis is necessary for the next step in glycolysis, which is another substrate level phosphorylation of ADP to form ATP.

= +1.7 kJ/mol

Reaction #10

Phosphoenolpyruvate + ADP + H+ <=> Pyruvate + ATP / See also HERE

Enzyme: Pyruvate Kinase
Notes - This reaction is important for several reasons. First, it generates ATP from the substrate-level phosporylation of ADP, putting the balance for glycolysis at a net gain of two molecules of ATP per molecule of glucose. Second, it is very favorable energetically, serving to "pull" the two preceding reactions (both of which have slightly positive values) forward. Third, the enzyme catalyzing the reaction, pyruvate kinase, is allosterically inactivated by ATP, alanine, and acetyl-CoA, allosterically activated by F1,6BP, and is inactivated by covalent modification (phosphorylation) from the kinase cascade.

= -31.4 kJ/mol

Anaerobic Metabolism of Pyruvate

Pyruvate is typically considered the last molecule produced in glycolysis. Under aerobic conditions pyruvate is transformed into acetyl-CoA, which then enters the citric acid cycle (next term) (see also HERE). Aerobic conditions provide a mechanism for converting NADH back to NAD+. NAD+ is essential for glycolysis to operate. Under anaerobic conditions, however, something else must be done to oxidize all the NADH formed in glycolysis (see HERE).

Key Points About Glycolysis

Entry of Other Sugars

Regulation of Glycolysis

Key regulatory enzymes in the glycolytic pathway include

A very important enzyme (called phosphofructokinase-2 or PFK2, but note that this is only part of this enzyme's activity) makes an allosteric regulator of glycolysis (F2,6BP) is NOT a glycolysis enzyme, but its product is a VERY potent regulator of glycolysis and gluconeogenesis (synthesis of glucose). As shown in Figure 16.19, PFK2 has a dual identity, with one part (the kinase part) capable of MAKING F2,6B and the other part (fructose-2,6 bisphosphatase - F2,6BPase) is capable of BREAKING DOWN F2,6BP. The kinase part of the enzyme is usually the part that is referred to as PFK2, while the other part is usually called F2,6BPase. Figure 16.20 shows the interconversion of the two forms. Basically, phosphorylation by protein kinase A activates the F2,6BPase while it inactivates the PFK2. Removal of the phosphate by phosphoprotein phosphatase reverses the activities, causing PFK2 to become active and the F2,6BPase to become inactive. This makes sense for the following reasons. First, when F2,6BPase is active, it degrades F2,6BP. Loss of F2,6BP leaves phosphofructokinase of glycolysis less active, slowing or stopping glycolysis. On the other hand, when PFK2 is active, it makes F2,6BP, which activates phosphofructokinase of glycolysis, and thus activates glycolysis. As you shall see below, F2,6BP has the OPPOSITE effects on gluconeogenesis. F2,6BP INHIBITS the gluconeogenesis enzyme, but in its absence, the enzyme is active.

Regulatory mechanisms for glycolysis include

Glucokinase

Glucokinase is an interesting liver form of hexokinase, called glucokinase. Glucokinase too catalyzes the conversion of glucose to glucose-6-phosphate using energy from ATP, but interestingly, glucokinase has a significantly lower affinity for glucose than hexokinase. This is very important for the liver, because this organ is involved in MAKING glucose for the rest of the body to use. If the glucose were made in the liver and the normal hexokinase were present, it would convert it back to G6P and it would never be released for the body to use.

Glucose Transporters

The lipid bilayer of the cellular membrane is an effective BARRIER to the movement of glucose across it. That would be a problem if there were not a means of transporting glucose into cells. Remember in multicellular organisms that glucose is made in specialized organs, such as the liver and and is transported by the bloodstream to desired tissues.

The job of moving glucose into cells is undertaken by specialized glucose transporters (GLUT proteins). Table 16.4 shows several glucose transporters and their tissue locations. The most common of these are GLUT1 and GLUT3, which are found in almost all mammalian cells.

Cancer and Glycolysis

Cancer cells have faster metabolic rates than normal cells and consequently must uptake and metabolize glucose faster than normal cells. Rapid metabolism leads to hypoxia (lack of oxygen compared to the need for it / see HERE). Cancer cells respond to hypoxia by activating a transcription factor called Hypoxia-Inducible Transcription Factor (HIF-1). HIF-1 acts as a transcription factor to increase the transcription and translation of most glycolytic enzymes and GLUT1 and GLUT3. Thus, these cells have both an increased supply of glucose and increased amounts of glycolytic enzymes to break it down. Remember that when oxygen is limiting, glycolysis is much less efficient, so to keep the energy levels high, cells must run more cycles of glycolysis. This is made possible by having increased transport and increased numbers of glycolytic enzymes.

Gluconeogenesis

Gluconeogenesis (HERE and HERE) is a central metabolic pathway involving biosynthesis of the sugar glucose. With the exception of four enzymes in gluconeogenesis that replace three enzymes in glycolysis, the enzymes of gluconeogenesis and glycolysis are the same. One starting point for gluconeogenesis is two molecules of pyruvate and the process ends with formation of one glucose molecule. The net effect of gluconeogenesis is the reversal of glycolysis. In gluconeogenesis, glucose is synthesized from smaller precursors. Now that you have learned about Delta G and values, 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 an excess of product (pyruvate) to reactant (glucose) that the cell could not generate. By the time the cell produced enough pyruvate and used its glucose by an attempted simple reversal, it would have starved to death for lack of glucose.

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. Though many of the steps of gluconeogenesis are the simple reversal of steps of glycolysis, there are three important steps in glycolysis that are replaced by four different steps in gluconeogenesis. Each of those steps is very energetically favored for glycolysis, so the gluconeogenesis pathway could not effectively reverse them. Instead, in two of the three steps, gluconeogenesis reactions omit regeneration of the ATP used in glycolysis, simply hydrolyzing the phosphate instead. This allows the reaction to proceed by "saving" the energy that would otherwise be put into regenerating ATP. The third glycolysis step that is bypassed is overcome by a set of two "sidestep" reactions in which ATP and GTP energy is expended to generate phosphoenolpyruvate, a high-energy intermediate.

Unique Gluconeogenesis Reactions

Figure 16.24 illustrates the pathway of gluconeogenesis. Seven enzymatic reactions are identical in glycolysis and gluconeogenesis. For gluconeogenesis, these seven enzymes simple catalyze the reversal of the reaction catalyzed for glycolysis. Three enzymes unique to glycolysis are replaced by four enzymes unique to gluconeogenesis. These enzymatic reactions are as follows:

1. Pyruvate + CO2 + H2O + ATP <=> Oxaloacetate + ADP + Pi + 2H+ (= -2.1 kJ/mol).
This reaction is catalyzed by pyruvate carboxylase (see HERE). Unlike the other reactions of glycolysis and gluconeogenesis, this reaction occurs in the mitochondrial matrix.

2. Oxaloacetate + GTP <=> Phosphoenolpyruvate + CO2 + GDP (=+2.9 kJ/mol)
This reaction is catalyzed by phosphoenolpyruvate carboxykinase - PEPCK.

3. Fructose-1,6-bisphosphate + H2O <=> Fructose-6-phosphate + Pi (=-16.3 kJ/mol)
This reaction is catalyzed by fructose 1,6 bisphosphatase.

4. Glucose-6-phosphate + H2O <=> Glucose + Pi ( = -12.1 kJ/mol)
This reaction is catalyzed by glucose-6-phosphatase (see HERE).

In all the other reactions, gluconeogenesis proceeds simply by reversing the corresponding reaction of glycolysis. The overall for gluconeogenesis is negative (-47.6 kJ/mol) thanks to expenditure of ATP energy. Glycolysis too has an overall negative (-73.3 kJ/mol) thanks to oxidation. The difference is that glycolysis accomplishes a negative while yielding reducing equivalents (2 NADH) and 2 net ATP, but the biosynthetic gluconeogenesis pathway requires use of 4 ATP and 2 GTP to achieve its overall negative .

All of the unique gluconeogenesis enzymes have important regulatory controls on them, as we shall see, because it is important that gluconeogenesis be turned turned off when glycolysis is turned on, and vice versa.

Other Gluconeogenesis Substrates

We often think of gluconeogenesis as starting with pyruvate, but other carbon sources than pyruvate can be used to make glucose via gluconeogenesis.

Note that breakdown products of fat metabolism (glycerol, propionyl-CoA), protein degradation (alanine, other amino acids), and anaerobic glycolysis (lactate) are substrates for gluconeogenesis. Notably, the primary breakdown product of fat, acetyl-CoA, cannot be effectively used by animals in gluconeogenesis. Some of the substrates are summarized as follows:

Reciprocal Regulation of Glycolysis and Gluconeogenesis

Anabolic (biosynthetic) pathways, such as gluconeogenesis and catabolic (biodegradative) pathways, such as glycolysis, have opposite needs and intentions. Gluconeogenesis creates glucose and glycolysis breaks it down. Gluconeogenesis uses ATP/GTP and glycolysis makes ATP. If glycolysis made as much ATP ad gluconeogenesis uses, the cell could at least break even if both processes occurred at the same time, but such efficiency is not possible. It takes more ATP to make glucose than is realized by breaking glucose down. Consequently, cells must be careful not to be running glycolysis and gluconeogenesis simultaneously or they will waste energy needlessly. This is true of any catabolic and related anabolic pathway (for example, fatty oxidation and fatty acid synthesis). Pathways like fatty acid oxidation and synthesis are spacially separated in the cells (oxidation in mitochondria and synthesis in cytoplasm), simplifying the separation.

Gluconeogenesis and glycolysis occur largely in the cytoplasm, with only the sythesis of oxaloacetate occurring in the mitochondrion. Consequently, spacial separaration is not sufficient for separately controlling these pathways. Instead, cells use a phenomenon known as reciprocal regulation (see HERE) which employs a mechanism to turn one of these pathways on and the same mechanism to turn off the other path (and vice-versa). This is accomplished by regulating the enzymes that are unique to each pathway. As shown in Figure 16.30, for example, the glycolysis enzyme, PFK, is activated by F2,6BP, but the corresponding enzyme in gluconeogenesis, fructose 1,6 bisphosphatase, is inhibited by F2,6BP. Other compounds reciprocally affecting glycolysis/gluconeogenesis enzymes include AMP and citrate. The most effective of all of these compounds is F2,6BP. Note also that ATP, which is an indicator of high energy is an inhibitor of glycolysis enzymes, but that AMP and ADP, which are indicators of low energy are inhibitors of gluconeogenesis enzymes. The enzyme PEPCK is notable for being controlled largely by the rate of its synthesis (trancriptionally and translationally).

Cori Cycle

With respect to energy, the liver and muscles are complementary organs. The liver is the major organ in the body for the synthesis of glucose. Muscles are major users of ATP. Figure 16.33 depicts the Cori cycle. In this cycle, actively exercising muscles generate lactate as a result of running glycolysis faster than the blood can deliver oxygen. As a consequence, the muscles go anaerobic and produce lactate. This lactate is of no use to muscle cells, so they dump it into the blood. Lactate travels in the blood to the liver, which takes it up and reoxidizes it back to pyruvate, catalyzed by the enzyme lactate dehydrogenase (note that the liver is close to the heart and lungs and has an abundant oxygen supply). Pyruvate in the liver is then converted to glucose by gluconeogenesis. The glucose thus made by the liver is dumped into the bloodstream where it is taken up by muscles and used for energy, completing the Cori cycle. Note that the enzyme lactate dehydrogenase has several different forms. One of the forms predominates in the heart and when this form of the enzyme is found in the blood, it is used as an indication of heart trouble, because heart tissue should not normally be going anaerobic.