Biochemistry, Aerobic Glycolysis

Fundamentals

Glycolysis occurs in the cytoplasm, where a 1 6-carbon glucose molecule is oxidized to generate 2 3-carbon pyruvate molecules. 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 ATP generation in the mitochondria. In cells with mitochondria, the pyruvate is decarboxylated by the pyruvate dehydrogenase complex to form acetyl-CoA, which feeds into the Tricarboxylic acid cycle and ultimately participates in ATP production.

During the absence of oxygen (anaerobic conditions) and in 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 10-step process begins with a molecule of glucose and ends with 2 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 traps the glucose molecule within the cell. Hexokinase has broad specificity and can phosphorylate all 6-carbon sugars, including glucose. In the liver and pancreatic beta cells, an isozyme of glucokinase exists and phosphorylates glucose exclusively.

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 by aldolase results in the formation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) 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: The interconversion of DHAP and glyceraldehyde-3-phosphate is catalyzed by triose phosphate isomerase. This isomerization yields 2 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, in which NAD+ is reduced to NADH, while the aldehyde group of glyceraldehyde-3-phosphate is oxidized to a carboxyl group, coupled with the attachment of a phosphate group. Limited cellular NAD+ levels require the reoxidation of NADH 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 increases under hypoxic conditions.

Step 8: Next, a reversible isomerization of 3-phosphoglycerate to 2-phosphoglycerate is catalyzed by phosphoglycerate mutase, in which the phosphate group is shifted from the third to the second carbon of phosphoglycerate. 

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

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

From here, pyruvate can follow an aerobic route to the mitochondria or an anaerobic route to form lactic acid. Irrespective of the path (aerobic or anaerobic) taken, glycolysis results in a net gain of 2 molecules of ATP per molecule of glucose.

Mechanism

The regulation of glycolysis occurs through covalent modification of rate-limiting enzymes, their allosteric activation or inhibition, and 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 the 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), 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, allowing the phosphatase form to remain active and decreasing F2,6-BP levels. This inhibits glycolysis, allowing gluconeogenesis to prevail.

Hormonal control plays an important role in regulating glycolysis. Carbohydrate consumption and its breakdown increase glucose levels and trigger the release of insulin, thereby increasing the insulin-to-glucagon ratio. Insulin activates glucokinase, PFK1, and pyruvate kinase, the 3 key enzymes that catalyze the irreversible steps of glycolysis to process the available substrate. At the same time, low glucagon levels inhibit gluconeogenesis. Long-term control through gene transcription is particularly important during fasting and starvation, and in diabetes, when the insulin-to-glucagon ratio is low. In such conditions, the synthesis of glucokinase, PFK1, and pyruvate kinase is decreased by modulation of gene transcription.[1][3]

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 2 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 the liver and pancreatic beta cells. Glucokinase has a much lower affinity for glucose than hexokinase and functions 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 glucose levels are low, high-affinity hexokinase binds glucose first, providing it to cells that need it most. Additionally, glucokinase in pancreatic beta cells acts as a glucose sensor, regulating the rate of glucose entry into cells and into glycolysis, thereby helping maintain proper blood glucose levels. 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 disease (COPD) are associated with an increase in the conversion of the glycolytic intermediate 1,3-BPG to 2,3-BPG by bisphosphoglycerate mutase. 2,3-BPG binds to deoxyhemoglobin with greater affinity than oxyhemoglobin and stabilizes it in its T-state. This allows oxygen to be released from deoxyhemoglobin, thereby increasing oxygen availability to cells. This is seen as a shift of the oxygen dissociation curve to the right.[9]

Pyruvate kinase deficiency: An 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 lack mitochondria; therefore, this enzyme deficiency can severely affect cells like RBCs, which rely on glycolysis as their sole fuel source. ATP is a precious commodity for RBCs and is required for the function of ATPase-dependent ion pumps that maintain membrane integrity. When compromised, it damages RBC membranes and causes hemolysis. This results in reduced oxygen delivery to tissues, leading to symptoms such as fatigue and shortness of breath. Hemolysis releases hemoglobin, whose breakdown ultimately increases bilirubin levels. Damage to the cell membrane results in distortion and loss of the smooth, biconcave structure, which appears 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, the 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, including embryonic and cancer cells. The survival of cancer cells depends on their ability to reprogram metabolic pathways to meet their needs. In normal cells with mitochondria, under aerobic conditions (presence of oxygen), pyruvate produced by glycolysis enters the mitochondria to participate in energy generation. Tumor cells differ in this regard, as they depend on aerobic glycolysis: in the absence of oxygen and mitochondrial function, pyruvate is diverted to lactate formation. This metabolic switch was first identified by Warburg and is known as the Warburg effect, which helps produce additional fuel for 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 can generate energy rapidly by diverting glucose to lactate rather than allowing it to follow its aerobic route through 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]