Introduction

Glycolysis is a central metabolic pathway that is used by all cells for the oxidation of glucose to generate energy in the form of ATP (Adenosine triphosphate) and intermediates for use in other metabolic pathways. Besides glucose, other hexose sugars such as fructose and galactose also end up in the glycolytic pathway for catabolism[1].

Fundamentals

Glycolysis occurs in the cytoplasm where one 6 carbon molecule of glucose is oxidized to generate two 3 carbon molecules of pyruvate. The fate of pyruvate depends on the presence or absence of mitochondria and oxygen in the cells. The electron transport chain is the major site of oxygen consumption and the generation of ATP in the mitochondria. In cells with mitochondria, the pyruvate is decarboxylated by pyruvate dehydrogenase complex to form Acetyl-CoA that feeds into the Tricarboxylic acid cycle and ultimately participates in ATP production.

During the absence of oxygen (anaerobic conditions) and in the cells lacking mitochondria, anaerobic glycolysis prevails. The pyruvate is reduced to lactate as NADH is reoxidized to NAD+ by lactate dehydrogenase. This process is an important source of ATP for cells that lack mitochondria, such as erythrocytes. During aerobic glycolysis, this NADH is transported by the malate aspartate shuttle or glycerol phosphate shuttle to the mitochondria, where it is reoxidized to NAD+ while it participates in the electron transport chain to produce ATP[1][2].

Cellular Level

Aerobic glycolysis is a series of reactions wherein oxygen is required to reoxidize NADH to NAD+, hence the name. This ten-step process begins with a molecule of glucose and ends up with two molecules of pyruvate[1].

Step 1: When a molecule of glucose enters the cell, it is immediately phosphorylated by the enzyme hexokinase to glucose-6-phosphate using the phosphate from the hydrolysis of ATP. This irreversible step serves to trap the glucose molecule within the cell. Hexokinase has broad specificity and can phosphorylate all six-carbon sugars, including glucose. In the liver and beta cells of the pancreas, an isozyme form glucokinase exists and solely phosphorylates glucose.

Step 2: Glucose-6-phosphate (aldose) is isomerized to fructose-6-phosphate (ketose) catalyzed by phosphoglucose isomerase. This reaction is readily reversible.

Step 3: Fructose-6-phosphate is phosphorylated to fructose-1, 6-bisphosphate by the enzyme phosphofructokinase-1 (PFK1). This is an irreversible, rate-limiting regulatory step. This committed step is the second ATP consuming step in glycolysis.

Step 4: Cleavage of fructose-1, 6-bisphosphate results in the formation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) by the enzyme aldolase in this unregulated, reversible reaction. Aldolase B, an isomer form in the liver, cleaves fructose-1-phosphate (in fructose metabolism) in addition to fructose-1, 6-bisphosphate.

Step 5: Interconversion of DHAP and glyceraldehyde-3-phosphate is carried out by triose phosphate isomerase. This isomerization results in producing of two molecules of glyceraldehyde-3-phosphate.

Step 6: Oxidation of glyceraldehyde-3-phosphate is catalyzed by glyceraldehyde-3-phosphate dehydrogenase and leads to the synthesis of 1, 3-bisphosphoglycerate. This is the first oxidation-reduction step in glycolysis where NAD+ is reduced to NADH, while the aldehyde group of glyceraldehyde -3-phosphate is oxidized to a carboxyl group coupled to the attachment of a phosphate group. Limited quantities of NAD+ in cells require the reoxidation of NADH back to NAD+. During aerobic conditions, NADH is reoxidized to NAD+ in the mitochondria, and during anaerobic conditions, it is regenerated by lactate dehydrogenase.

Step 7: Formation of 3-phosphoglycerate from 1,3-bisphosphoglycerate (1,3-BPG) is the first ATP generating step in glycolysis. The phosphate group attached during the formation of 1,3-BPG in the previous step is used to phosphorylate ADP with the help of phosphoglycerate kinase, thereby generating ATP. This substrate-level phosphorylation generates 2 ATPs. Some of the 1,3-BPG is also converted to 2,3-bisphosphoglycerate (2,3-BPG) by bisphosphoglycerate mutase, an important product that helps oxygen delivery to cells. Normally 2,3-BPG is present in trace quantities, but its production will increase during hypoxic conditions.

Step 8: Next, a reversible isomerization reaction of 3-phosphoglycerate to 2-phosphoglycerate is carried out by phosphoglycerate mutase, where the phosphate group is shifted from the third carbon to the second carbon of phosphoglycerate. 

Step 9: 2-phosphoglycerate is converted to phosphoenolpyruvate, which contains the high-energy enol phosphate.

Step 10: The final step in glycolysis is the enzymatic conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase. Substrate level phosphorylation occurs in this irreversible step to generate 2 molecules of ATP.

From here, the pyruvate can go through an aerobic route to the mitochondria or anaerobic route to form lactic acid. Irrespective of the path (aerobic or anaerobic) taken, glycolysis results in a net gain of two molecules of ATP per molecule of glucose.

Mechanism

Mechanism of regulation of glycolysis occurs through covalent modification of rate-limiting enzymes, their allosteric activation or inhibition, and by hormonal control.

A bifunctional enzyme PFK2/Fructose bisphosphatase, with kinase and phosphatase activity, is an important player in allosteric regulation. F2,6-BP is an allosteric effector whose concentration depends on the ratio of insulin and glucagon. PFK1 is positively regulated by F2,6-BP, whose synthesis is catalyzed by kinase activity of phosphofructokinase-2 (PFK-2). When there are plenty of substrates available, high levels of insulin activate a protein phosphatase that dephosphorylates phosphofructokinase-2 (PFK2), making it active, thereby promoting glycolysis.

On the other hand, when glucagon levels are high, a rise in cAMP activates protein kinase A which favors the phosphorylated form of the bifunctional enzyme. Phosphorylation inactivates PFK2 and allows the phosphatase form to stay active, causing the levels of F2,6-BP to decrease. This inhibits glycolysis, allowing gluconeogenesis to prevail.

Hormonal control plays an important role in the regulation of glycolysis. Carbohydrate consumption and its breakdown lead to an increase in the levels of glucose and trigger the release of insulin, resulting in an increase in the ratio of insulin to glucagon. Insulin activates glucokinase, PFK1, and pyruvate kinase, the three important enzymes catalyzing the irreversible steps in glycolysis in order to process the available substrate. At the same time, low glucagon levels ensure that gluconeogenesis is inhibited. Long-term control through gene transcription is particularly important in fasting and starvation states and in diabetes when the ratio of insulin to glucagon is low. In such conditions, the synthesis of glucokinase, PFK1, and pyruvate kinase are decreased by modulation of gene transcription[1][3][1].

Clinical Significance

Glucokinase Deficiency: Both glucokinase and hexokinase perform the same function of phosphorylating glucose to glucose-6-phosphate and trapping it in the cell. The difference between the two lies in their location and affinity to glucose. Glucokinase is present in the liver and pancreatic beta cells. Hexokinase, its isomer form, is present in tissues other than liver and pancreatic beta cells. Glucokinase has a much lower affinity for glucose than hexokinase and will function only when the glucose levels are high. After a meal, when blood glucose levels rise, glucokinase directs it toward glycogen synthesis and storage in the liver. When the glucose levels are low, hexokinase with high affinity will get to the glucose first so as to provide the glucose to cells that need it the most. Additionally, glucokinase in pancreatic beta cells acts as a glucose sensor and regulates the rate of entry of glucose into cells and into glycolysis, and therefore helps maintain the proper glucose levels in the blood. Heterozygous inactivating mutations of glucokinase result in maturity-onset diabetes of the young type 2 (MODY2 or GCK-MODY)[4][5]. Homozygous mutations result in a complete deficiency of this enzyme and cause neonatal diabetes mellitus[6][7][8]. 

2,3-Bisphosphoglycerate:  Human RBCs normally have low levels of 2,3-BPG. During decreased availability of oxygen, as in high altitudes, respiratory diseases such as asthma, or chronic obstructive pulmonary diseases (COPD), there is an increase in the conversion of the glycolytic intermediate 1,3-BPG to 2,3-BPG by the action of bisphosphoglycerate mutase. 2,3-BPG binds to deoxyhemoglobin with greater affinity than oxyhemoglobin and stabilizes it in its T-state. This allows the oxygen to unload from the deoxyhemoglobin, thus increasing the oxygen availability to the cells. This is seen as a shift of the oxygen dissociation curve to the right[9].

Pyruvate kinase deficiency: Autosomal recessive disorder of pyruvate kinase deficiency occurs due to mutations in the PKLR gene. Pyruvate kinase catalyzes the final irreversible step towards the formation of pyruvate while producing ATP. Mature RBCs do not have mitochondria, and therefore, this enzyme deficiency can severely impact cells like RBCs, where glycolysis is the sole fuel source. ATP is a precious commodity for RBCs and is required for the functioning of the ATPase-dependent ion pumps to maintain membrane integrity. When compromised, it results in damage to membranes of RBCs and causes hemolysis. This results in a reduction of oxygen delivery to tissues manifesting symptoms such as fatigue and shortness of breath. Hemolysis releases the hemoglobin, whose breakdown ultimately results in increased levels of bilirubin. Damage to the cell membrane results in distortion and loss of smooth biconcave structure and is seen as thorny projections. These spiculated appearing RBCs are called echinocytes. A decrease in the number of RBCs prompts the appearance of immature RBCs or reticulocytes, a feature typically seen in pyruvate kinase deficiency.  However, deficiency of the isozyme form of pyruvate kinase in the hepatocytes does not show any effect, as the presence of mitochondria allows for the generation of ATP. The 2,3-BPG levels are subsequently elevated as a compensatory mechanism to increase oxygen delivery to the cells, although its synthesis does not produce ATP[10].

Role of Pyruvate kinase in Cancer:

Pyruvate kinase has been shown to be upregulated in highly proliferating cells such as embryonic cells and cancer cells. The survival of cancer cells is dependent on their ability to reprogram the metabolic pathways to suit their needs. In normal cells, with mitochondria, under aerobic conditions (presence of oxygen), pyruvate produced from glycolysis enters the mitochondria to participate in the process of energy generation. Tumor cells are different in this regard as they are dependent on aerobic glycolysis, wherein the presence of oxygen and the availability of mitochondria, the pyruvate, is diverted to the formation of lactate. This metabolic switch was first identified by Warburg and is known as the Warburg effect, which helps the production of additional fuel for the cancer cells in the form of lactate. An M2 isoform of pyruvate kinase has been shown to be upregulated in cancer cells[11][12][13].

So far, it is unclear as to why the cancer cells exhibit enhanced aerobic glycolysis. It is hypothesized that cancer cells are able to generate energy rapidly by diverting glucose to form lactate rather than letting glucose go through its aerobic route to the TCA cycle and electron transport chain. Other proposed mechanisms suggest the use of aerobic glycolysis by tumor cells increases signal transduction, increases the flux towards biosynthetic pathways, and, finally, the generation of lactate creates an acidic microenvironment more conducive to invasiveness and metastasis [14][15][16].