Glycolysis

Glycolysis is a series of biochemical reactions by which a molecule of glucose (Glc) is oxidized to two molecules of pyruvic acid (Pyr).

The word glycolysis is from Greek glyk (meaning sweet) and lysis (meaning dissolving). It is the initial process of many pathways of carbohydrate catabolism, and serves two principal functions: generation of high-energy molecules (ATP and NADH), and production of a variety of six- or three-carbon intermediate metabolites, which may be removed at various steps in the process for other intracellular purposes (such as nucleotide biosynthesis).

Glycolysis is one of the most universal metabolic processes known, and occurs (with variations) in many types of cells in nearly all types of organisms. Glycolysis alone produces less energy per glucose molecule than complete aerobic oxidation, and so flux through the pathway is greater in anaerobic conditions (i.e., in the absence of oxygen).

The most common and well-known type of glycolysis is the Embden-Meyerhof pathway, initially elucidated by Gustav Embden and Otto Meyerhof. The term can be taken to include alternative pathways, such as the Entner-Doudoroff Pathway. However, glycolysis will be used here as a synonym for the Embden-Meyerhof pathway.

Overview
The overall reaction of glycolysis is:


 * Glc + 2 NAD+ + 2 ADP + 2 Pi &#8594; 2 NADH + 2 Pyr + 2 ATP + 2 H2O + 2 H+

So, for simple fermentations, the metabolism of 1 molecule of glucose has a net yield of 2 molecules of ATP. Cells performing respiration synthesize much more ATP, but this is not considered part of glycolysis proper, although these aerobic reactions do use the product of glycolysis. Eukaryotic aerobic respiration produces an additional 34 molecules (approximately) of ATP for each glucose molecule oxidized. Unlike most of the molecules of ATP produced via aerobic respiration, those of glycolysis are produced by substrate-level phosphorylation.

In eukaryotes, glycolysis takes place within the cytosol of the cell. Some of the glycolytic reactions are conserved in the Calvin cycle that functions inside the chloroplast. This is consistent with the fact that glycolysis is highly conserved in evolution, being common to nearly all living organisms. This suggests great antiquity; it may have originated with the first prokaryotes, 3.5 billion years ago or more.

Preparatory phase
The first five steps are regarded as a preparatory phase since they actually consume energy as the glucose is converted to two three-carbon sugars phosphates (G3P). The bold abbreviations in the two tables correspond to the nomenclature used in the diagram.

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



Entry of sugars
The first step in glycolysis is phosphorylation of Glc by a family of enzymes called HKs to form G6P. In the liver, an isozyme of hexokinase called GCK is used, which differs primarily in regulatory properties. This reaction consumes 1 ATP, but the energy is well-spent - it keeps [Glc] i low as to allow continuous entry of Glc through its plasma membrane transporters; prevents Glc leakage out - the cell lacks such transporters for G6P; activates Glc preparing it for the next metabolic changes.

G6P is then rearranged into F6P by GPI. Fru can also enter the glycolytic pathway via phosphorylation at this point.

Control of flux
The flux through the glycolytic pathway must be adjusted in response to conditions both inside and outside the cell. The rate is 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 hexokinase, phosphofructokinase, and pyruvate kinase are effectively irreversible. In metabolic pathways, such enzymes are potential sites of control, and all these three enzymes serve this purpose in glycolysis.

There are several different ways to regulate the activity of an enzyme. An immediate form of control is feedback via allosteric effectors or by covalent modification. A slower form of control is transcriptional regulation that controls the amounts of these important enzymes.

Phosphofructokinase-1
Phosphofructokinase is an important control point in the glycolytic pathway since it is immediately downstream of the entry points for hexose sugars.

High levels of ATP inhibit the PFK enzyme by lowering its affinity for F6P. ATP causes this control by binding to a specific regulatory site that is distinct from the catalytic site. This is a good example of allosteric control. AMP can reverse the inhibitory effect of ATP. A consequence is that PFK is tightly controlled by the ratio of ATP/AMP in the cell. This makes sense since these molecules are direct indicators of the energy charge in the cell.

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

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).

Fructose 2,6-bisphosphate (F2,6BP) is a potent activator of phosphofructokinase (PFK-1) that is synthesised when F6P is phosphorylated by a second phosphofructokinase (PFK2). This second enzyme is inactive when cAMP is high, and links the regulation of glycolysis to hormone activity in the body. Both glucagon and adrenalin cause high levels of cAMP in the liver. The result is lower levels of liver fructose 2,6-bisphosphate such that gluconeogenesis (glycolysis in reverse) is favored. This is consistent with the role of the liver in such situations since the response of the liver to these hormones is to releases glucose to the blood.

Energy pay-off
Each molecule of GADP is then oxidized by a molecule of NAD+ in the presence of GAP, forming 1,3-bisphosphoglycerate. In the next step, PGK generates a molecule of ATP while forming 3-phosphoglycerate. At this step, glycolysis has reached the break-even point: 2 molecules of ATP were consumed, and 2 new molecules have been synthesized. This step, one of the two substrate-level phosphorylation steps, requires ADP; thus, when the cell has plenty of ATP (and little ADP) this reaction does not occur. Because ATP decays relatively quickly when it is not metabolized, this is an important regulatory point in the glycolytic pathway. PGAM then forms 2-phosphoglycerate; ENO then forms phosphoenolpyruvate; and another substrate-level phosphorylation then forms a molecule of Pyr and a molecule of ATP by means of the enzyme PK. This serves as an additional regulatory step.

After the formation of F1,6bP, many of the reactions are energetically unfavorable. The only reactions that are favorable are the 2 substrate-level phosphorylation steps that result in the formation of ATP. These two reactions pull the glycolytic pathway to completion.

Follow-up
The ultimate fate of pyruvate and NADH produced in glycolysis depends upon the organism and the conditions, most notably the presence or absence of oxygen and other external electron acceptors.

In aerobic organisms, pyruvate typically enters the mitochondria where it is fully oxidized to carbon dioxide and water by pyruvate decarboxylase and the set of enzymes of the citric acid cycle (also known as the TCA or Krebs cycle). The products of pyruvate are sequentially dehydrogenated as they pass through the cycle conserving the hydrogen equivalents via the reduction of NAD + to NADH. NADH is ultimately oxidized by an electron transport chain using oxygen as final electron acceptor to produce a large amount of ATP via the action of the ATP synthase complex, a process known as oxidative phosphorylation. A small amount of ATP is also produced by substrate-level phosphorylation during the TCA cycle.

Although human metabolism is primarily aerobic, under hypoxic (or partially anaerobic) conditions, for example in overworked muscles that are starved of oxygen or in infarcted heart muscle cells, pyruvate is converted to the waste product lactate. This and similar reactions are known as fermentation, and they are a solution to maintaining the metabolic flux through glycolysis in response to an anaerobic or severely hypoxic environment.

Although fermentation does not produce much energy, it is critical for an anaerobic or hypoxic cell, since it regenerates NAD + that is required for glycolysis to proceed. This is important for normal cellular function, as glycolysis is the only source of ATP in anaerobic or severely hypoxic conditions.

There are several types of fermentation wherein 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, whereas yeast produces ethanol and carbon dioxide. Anaerobic bacteria are capable of using a wide variety of compounds, other than oxygen, as terminal electron acceptors in respiration: nitrogenous compounds (such as nitrates and nitrites), sulphur compounds (such as sulphates, sulphites, sulphur dioxide, and elemental sulphur), carbon dioxide, iron compounds, manganese compounds, cobalt compounds, and uranium compounds.

Intermediates for other pathways
This article concentrates on the catabolic role of glycolysis with regard to converting potential chemical energy to usable chemical energy during the oxidation of glucose to pyruvate. Many of the metabolites in the glycolytic pathway are also used by anabolic pathways, and, as a consequence, flux through the pathway is critical also to maintain a supply carbon skeletons for biosynthesis.

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 a role 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.

High aerobic glycolysis
During anaerobic conditions, glycolysis is the cellular mechanism to obtain ATP, by fermentation. However, in mammalian cells, glycolysis is coupled with aerobic respiration. In the presence of oxygen, mitochondria take up pyruvate, the end-product of glycolysis, and further oxidize it into CO2 and water. As a result, the flux through the glycolytic pathway is lower during aerobic conditions since the full oxidation of one molecule of pyruvate (equivalent to one-half molecule of glucose) can lead to 18 times more ATP. Malignant rapidly-growing tumor cells, however, have glycolytic rates that are up to 200 times higher than that 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 the high glycolytic rate in tumor cells. Nevertheless, there is also strong experimental evidence that attributes these high aerobic glycolytic rates to an overexpressed form of mitochondrially-bound hexokinase responsible for driving the high glycolytic activity when oxygen is not necessarily depleted. This phenomenon was first described in 1930 by Otto Warburg, and hence it is referred to as the Warburg Effect. This has a current important medical application, as aerobic glycolysis by malignant tumors is utilized clinically to diagnose and monitor treatment responses of cancers by imaging uptake of 2-18F-2-deoxyglucose (a radioactive modified hexokinase substrate) with positron emission tomography (PET),.

Alternative nomenclature
Some of the metabolites in glycolysis have alternative names and nomenclature. In part, this is because some of them are common to other pathways, such as the Calvin cycle.