Glycolysis

Glycolysis is a series of biochemical reactions by which one molecule of glucose (Glc) is oxidized to two molecules of pyruvic acid (Pyr) and a relatively small amount of the universal energy storage molecule adenosine triphosphate (ATP). This breakdown of the simple sugar glucose serves three principal functions:

• Generation of the high-energy molecules (ATP and NADH), which are used as cellular energy sources in both aerobic respiration (with oxygen) and anaerobic respiration (without oxygen)
• Production of pyruvate for the citric acid cycle as part of aerobic respiration
• Production of a variety of six- or three-carbon intermediate metabolites, which may be removed at various steps in the process for other cellular purposes (such as nucleotide biosynthesis).

As the foundation of both aerobic and anaerobic respiration, glycolysis is the archetype of universal metabolic processes known and occurring (with variations) in many types of cells in nearly all organisms. Glycolysis, through anaerobic respiration, is the main energy source in many prokaryotes, eukaryotic cells devoid of mitochondria (e.g., mature erythrocytes), and eukaryotic cells under low-oxygen conditions (e.g., heavily-exercising muscle or fermenting yeast). The near ubiquity of these reactions suggests harmony and connectivity among organisms and great antiquity; glycolysis may have originated with the first prokaryotes (organisms without a cell nucleus) at least 3.5 billion years ago.

For organisms that live in the absence of oxygen (obligate anaerobes) or that can live either in the presence or absence of oxygen (facultative anaerobes), glycolysis serves as the principle means of oxidizing glucose for chemical energy.

However, for aerobic organisms such as humans, glycolysis is only the initial stage of carbohydrate catabolism; the end-products of glycolysis enter into the citric acid cycle (also known as the TCA or Krebs cycle) and the electron transport chain for further oxidation. These pathways together produce considerably more energy per glucose molecule than anaerobic oxidation. However, there are times when humans rely on glycolysis for fuel—for example, during short periods of intense exertion, the muscle cells may switch to glycolysis when oxygen is depleted. Though less energy efficient, glycolysis produces energy at a rate 100 times faster than aerobic respiration.

Increased activity in the glycolytic pathway can also be an indicator of disease in humans. Malignant, rapidly-growing tumor cells have glycolytic rates that are up to 200 times higher than those of their normal tissues of origin.

Overview

In eukaryotes (organisms in which genetic material is stored in a membrane-bound nucleus), glycolysis takes place within the cytosol, or the internal fluid of the cell. Glucose is made available for entry into the reaction pathway through the breakdown of polysaccharides, such as the glycogen stored in the liver or muscle, or the starch ingested in the diet, by digestive enzymes. In animals, the liberated simple sugars (monosaccharides) next travel from the small intestine into the bloodstream, which carries them to the cells of the liver and other energy-hungry tissues.

The overall reaction of glycolysis can be expressed as follows:

$Glc+2NAD^{+}+2ADP+2P_{i}\to 2NADH+2Pyr+2ATP+2H_{2}O+2H^{+}$

The reactions of glycolysis

The glycolytic pathway is typically thought of as occurring in two phases:

1. In the preparatory phase (reactions 1-5), 2 ATP are invested in order to prepare the glucose molecule for further catabolism.
2. In the energy-payoff phase (reactions 6-10), the initial energy invested is recouped, and an additional 2 ATP are generated.

The most common and well-studied type of glycolysis is the Embden-Meyerhof pathway, initially elucidated by Gustav Embden and Otto Meyerhof; although alternative pathways have been described, glycolysis will be used here as a synonym for the Embden-Meyerhof pathway.

Energy-investment phase

The first five steps of glycolysis (described in the table below) prepare glucose for breakdown by "trapping" it in the cell and destabilizing it, which requires an investment of 2 ATP. In this first phase, glucose, a six-carbon molecule, is converted to two three-carbon sugars (Glyceraldehyde 3-phosphate—G3P).

Reaction	Substrate		Enzyme		Enzyme class	Comment
1	glucose	Glc	hexokinase	HK	transferase	One ATP (per molecule of glucose) is invested in this first step, in which the alcohol group in the glucose molecule reacts with a terminal phosphate group of ATP. The energy is well-spent—it keeps glucose levels low to allow continuous entry of Glc into the cell and prevents Glc leakage out, as the cell lacks membrane transporters for G6P. Because this reaction has a highly negative change in free energy, this step is irreversible.
2	glucose-6-phosphate	G6P	phosphoglucose isomerase	PGI	isomerase	G6P is then rearranged into Fructose 6-phosphate (F6P). Fructose (Fru) can also enter the glycolytic pathway at this point. The change in structure is observed through a redox reaction, in which the aldehyde has been reduced to an alcohol, and the adjacent carbon has been oxidized to form a ketone. While this reaction is not normally favorable, it is driven by a low concentration of F6P, which is constantly consumed during the next step of glycolysis. (This phenomenon can be explained through Le Chatelier's Principle.)
3	fructose 6-phosphate	F6P	phosphofructokinase	PFK-1	transferase	The energy expenditure of a second ATP in this step is justified in two ways: the glycolytic process (up to this step) is now irreversible, and the energy supplied destabilizes the molecule, preparing it for breakdown.
4	fructose 1,6-bisphosphate	F1,6BP	aldolase	ALDO	lyase	Destabilizing the molecule in the previous reaction allows the hexose ring to be split by ALDO into two triose sugars, DHAP and GADP.
5	dihydroxyacetone phosphate	DHAP	triose phosphate isomerase	TPI	isomerase	TPI rapidly inter-converts DHAP with glyceraldehyde 3-phosphate (GADP), which proceeds into the next stages of glycolysis.

Energy-payoff phase

The second half of glycolysis is known as the payoff phase; it is characterized by a net gain of the energy-rich molecules ATP and NADH. Since the breakdown of glucose generates two triose sugars in the preparatory phase, each reaction in the pay-off phase occurs twice per glucose molecule. This phase yields 2 NADH molecules and 4 ATP molecules, leading to a net gain of 2 NADH molecules and 2 ATP molecules from the glycolytic pathway per glucose.

The mechanism by which ATP molecules are generated in glycolysis is known as substrate-level phosphorylation: a phosphoryl group is transferred from a glycolytic intermediate to ADP by an enzyme called a kinase.

Reaction	Substrate		Enzyme		Enzyme class	Comment
6	glyceraldehyde 3-phosphate	GADP	glyceraldehyde 3-phosphate dehydrogenase	GAP	oxidoreductase	The triose sugars are dehydrogenated, and inorganic phosphate is added. The hydrogen is used to reduce two molecules of NAD, a hydrogen carrier, to yield NADH+H+.
7	1,3-bisphosphoglycerate	1,3BPG	phosphoglycerate kinase	PGK	transferase	This reaction converts ADP to ATP by an enzymatic transfer of a phosphate to ADP; it is an example of substrate-level phosphorylation. The 2 ATP invested in the preparatory phase are recovered at this point.
8	3-phosphoglycerate	3PG	phosphoglyceromutase	PGAM	mutase
9	2-phosphoglycerate	2PG	enolase	ENO	lyase
10	phosphoenolpyruvate	PEP	pyruvate kinase	PK	transferase	This reaction is another example of substrate-level phosphorylation; it converts ADP, the discharged form of ATP, into a charged ATP molecule, forming pyruvate (Pyr).

Regulation of glycolysis

The flux, or rate of flow through the glycolytic pathway, may be regulated to meet two major cellular needs: (1) the production of ATP, and (2) the provision of building blocks for biosynthetic reactions.

In glycolysis, the reactions catalyzed by the enzymes hexokinase, phosphofructokinase, and pyruvate kinase are effectively irreversible; in metabolic pathways, such enzymes are potential sites of control.

Hexokinase

Hexokinase is inhibited by glucose-6-phosphate (G6P), the product it forms in the first step of glycolysis (an example of feedback inhibition). The control of hexokinase is necessary to prevent an accumulation of G6P in the cell when flux through the glycolytic pathway is low. Although glucose will continue to enter the cell, it can readily diffuse back into the blood when hexokinase is inactive. If hexokinase remained active during low glycolytic flux, the G6P would accumulate, and the extra solute would cause the cells to enlarge due to osmosis.

In liver cells, the enzyme glucokinase rather than hexokinase catalyzes the phosphorylation of glucose to G6P—an important distinction that allows the liver to maintain stable blood sugar levels. In liver cells, excess G6P is stored as glycogen, which can be made available during times of fasting, as brain cells depend on glucose as an energy source. Unlike hexokinase, glucokinase is not inhibited by high levels of G6P, so that extra glucose molecules can be converted to G6P to be stored as glycogen. During times of hypoglycemia (low blood sugar), the glycogen can be converted back to G6P and then to glucose.

Phosphofructokinase

Phosphofructokinase (PFK) is the most important control point in the glycolytic pathway of mammals since it catalyzes the reaction immediately following the entry of 6-carbon sugars such as glucose and fructose.

High levels of ATP inhibit the PFK enzyme by lowering its affinity for F6P. ATP achieves this inhibition through the mechanism of allosteric control; i.e., it binds to a specific regulatory site (the allosteric site) that is distinct from the catalytic site (or active site). Since AMP can reverse the inhibitory effect of ATP, PFK is tightly controlled by the ratio of ATP/AMP in the cell. These molecules are direct indicators of the energy charge in the cell: so, in essence, glycolysis increases as the energy charge falls.

Since glycolysis is also a source of carbon skeletons for biosynthesis, a feedback control from the carbon skeleton pool, signaling that biosynthetic precursors are abundant, is useful. Citrate, an early intermediate in the citric acid cycle, is an example of a metabolite that regulates phosphofructokinase by enhancing the inhibitory effect of ATP.

Low pH also inhibits phosphofructokinase activity and prevents the excessive rise of lactic acid during anaerobic conditions that could otherwise cause a drop in blood pH (acidosis), a potentially life threatening condition.

Fructose 2,6-bisphosphate (F2,6BP) is a potent activator of phosphofructokinase (PFK-1) that is synthesized when F6P is phosphorylated by a second phosphofructokinase (PFK-2). This second enzyme is inactive when the level of AMP in the cell is high.

Pyruvate kinase

Pyruvate kinase (PK) activity catalyzes the final step of glycolysis, in which pyruvate is formed. The enzyme is activated by fructose 1,6-bisphosphate, an intermediate in glycolysis, driving the rate of glycolysis when more substrate is present.

The inhibition of PK by ATP is similar to the effect of ATP on PFK-1. The binding of ATP to the inhibitor site reduces its affinity for PEP. The liver enzyme is also controlled at the level of synthesis. Increased carbohydrate ingestion induces the synthesis of PK, resulting in elevated cellular levels of the enzyme. This slower form of control, which responds to long-term dietary and environmental factors, is referred to as transcriptional regulation.

Next steps

The ultimate fate of the pyruvate and NADH produced in glycolysis depends upon the individual organism and the specific cellular conditions, most notably the presence or absence of oxygen. In order for glycolysis to proceed, NADH must donate either an electron or a proton (hydrogen) to an acceptor molecule (either oxygen or another organic molecule), allowing NADH to re-enter the pathway as NAD⁺.

Aerobic respiration

In aerobic organisms, pyruvate typically moves from the cytosol into the mitochondria of the cell, where it is fully oxidized to carbon dioxide and water by pyruvate decarboxylase and the enzymes of the citric acid cycle. NADH is ultimately oxidized by an electron transport chain, using oxygen as the final electron acceptor to produce a large amount of ATP through a process known as oxidative phosphorylation. Aerobic respiration produces an additional 34 molecules (approximately) of ATP for each glucose molecule oxidized.

Fermentation

Although human metabolism is primarily aerobic, in the partial or complete absence of oxygen (for example, in overworked muscles that are starved of oxygen or in infarcted heart muscle cells), pyruvate can be converted to the waste product lactate. (The lactic-acid buildup in our muscles causes "the burn" we associate with intense exercise.) This reaction, which is an example of a fermentation, is a solution to maintaining the metabolic flux through glycolysis in the absence of oxygen or when oxygen levels are low.

There are several types of fermentations in which pyruvate and NADH are anaerobically metabolized to yield any of a variety of products with an organic molecule acting as the final hydrogen acceptor. For example, the bacteria involved in making yogurt simply reduce pyruvate to lactic acid. In organisms such as brewers' yeast, a carboxyl group is first removed from pyruvate to form acetaldehyde and carbon dioxide; the acetaldehyde is then reduced to yield ethanol and NAD⁺. Anaerobic bacteria are capable of using a wide variety of compounds other than oxygen as terminal electron acceptors.

Synthesis of intermediates for other pathways

In addition to the important catabolic role of glycolysis, many of the metabolites in the glycolytic pathway are also used by anabolic pathways (such as the pentose phosphate pathway, which serves to generate 5-carbon sugars). As a consequence, flux through the pathway is critical to maintain a supply of carbon skeletons for biosynthesis of other molecules.

From an energy perspective, NADH is either recycled to NAD+ during anaerobic conditions (to maintain the flux through the glycolytic pathway), or used during aerobic conditions to produce more ATP by oxidative phosphorylation. From an anabolic metabolism perspective, the NADH has an additional function to drive synthetic reactions, doing so by directly or indirectly reducing the pool of NADP+ in the cell to NADPH, which is another important reducing agent for biosynthetic pathways in a cell.

Glycolysis as an indicator of disease

The glycolytic rates in malignant, rapidly-growing tumor cells are up to 200 times higher than those of their normal tissues of origin, despite the ample availability of oxygen. A classical explanation holds that the local depletion of oxygen within the tumor is the cause of increased glycolysis in these cells. However, there is also strong experimental evidence that attributes these high rates to an over-expressed form of the enzyme hexokinase (Bustamante and Pedersen 2005),which is responsible for driving the high glycolytic activity when oxygen is not necessarily depleted. This finding currently has an important medical application: aerobic glycolysis by malignant tumors is utilized clinically to diagnose and monitor treatment responses of cancers using medical imaging techniques (Pauwels et al. 2000, PETNET Solutions 2006).

References

ISBN links support NWE through referral fees

• Bustamante, E., and P. L. Pedersen. 1977. High aerobic glycolysis of rat hepatoma cells in culture: Role of mitochondrial hexokinase. Proceedings of the National Academy of Sciences 74(9):3735-3739. (http://www.pnas.org/cgi/reprint/74/9/3735)
• Pauwels, E. K. J., E. J. C. Sturm, E. Bombardier, F. J. Cleton, and M. P. M. Stokkel. 2000. Positron-emission tomography with ¹⁸Flourodeoxyglucose. J. Cancer Res. Clin. Orcol. 126:549-559.
• PETNET Solution. 2006. Do I Have Cancer?. (accessed August 25, 2006).
• Stryer, Lubert. 1987. Biochemistry, 3rd edition. New York, NY: W.H. Freeman.

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