Divyan Bavan
Introduction
Metabolism is one of the main characteristics for any form of life. This process consists of many pathways, each maintaining a balance between energy usage and creation. Since this balance is critical to the organism’s existence, it is no surprise that these pathways are heavily regulated. Glycolysis is no exception. This pathway catalyzes the conversion of glucose into pyruvate, producing two molecules of ATP and NADH in the process. This reaction consists of ten steps, each with specific enzymes: phosphofructokinase, hexokinase, pyruvate kinase, and more. While it is one of the oldest pathways, many of the mechanisms used to regulate glycolysis are found throughout an organism’s metabolism. These regulatory features—allosteric control, hormone action, and transcriptional regulation—are critical to its control. By understanding these mechanisms and how they relate to other forms of glucose metabolism, it is possible to form an integrated picture of metabolic control.
Allosteric Control
Enzymatic activity is one of the core components of metabolic activity. By controlling the rate at which enzymes catalyze a reaction, it is possible to regulate the speed and flux of a reaction. This is demonstrated throughout metabolism. Chemicals—often the products or intermediates of the pathway—can bind to allosteric sites on enzymes and alter their activity. In glycolysis, the enzymes controlled in this manner are hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK). These enzymes catalyze the slowest steps of glycolysis, which function far from equilibrium. This means that they have the largest control over total glycolytic flux and are critical to regulate (Voet and Voet, 2011).
Hexokinase Control
HK catalyzes the conversion of glucose into glucose-6-phosphate (G6P). This step traps glucose within the cell, committing it to further metabolism. Furthermore, this reaction is highly exergonic and thus, irreversible. By altering the activity of HK, it is possible to control the total amount of glucose being processed. This occurs through negative feedback; G6P directly inhibits HK. This stabilizes the amount of glucose being processed at once (Voet and Voet, 2011).
Phosphofructokinase Control
PFK-1 is the primary control point for glycolysis. This enzyme is responsible for converting fructose-6-phosphate (F6P) and ATP to fructose-1,6-bisphosphate (FBP). Like hexokinase, this step is irreversible and highly exergonic. However, PFK-1 is a larger control point as it is the first committed step of glycolysis. Prior to this step, glucose can take alternate paths such as the pentose phosphate or glycogen synthesis. Therefore, controlling the activity of PFK-1 is critical for determining the overall glycolytic flux.
PFK has several activators and inhibitors. For example, one inhibitor of PFK is ATP: the main energy store for the cell. As one of the products of glycolysis—and cellular respiration as a whole—it signals whether more energy is needed. In the case where ATP is plentiful and glycolysis is not needed, ATP downregulates PFK’s activity. The mechanism for this lies in PFK’s structure. PFK can take two forms: an R and T state. The R state is more catalytically active as it has a higher affinity for F6P. As a substrate, ATP binds to both states equally. However, in excess, ATP binds to an inhibitory site on the PFK subunit; this interaction is more stable in the T state. Therefore, ATP binding to the inhibitory site leads to more PFK taking the T state, reducing glycolytic flux. Other inhibitors of PFK such as PEP and citrate—which are also downstream products of cellular respiration—act with similar logic, reducing PFK output as a response to product accumulation (Voet and Voet, 2011)
The opposite is true for activators of PFK. These chemicals signal the need to break down glucose and thus, increase PFK activity. Some examples of these activators are AMP, ADP, fructose-2,6-bisphosphate (F2,6BP). These chemicals essentially reverse the effect of ATP inhibition, stabilizing the R state and increasing glycolytic flux (Voet and Voet, 2018). While these chemicals can be controlled internally through energy deprivation responses, they can also be controlled hormonally. This is the case with F2,6BP, and will be discussed later (Voet and Voet, 2011).
Pyruvate Kinase Control
PK catalyzes the last reaction of glycolysis, converting phosphoenolpyruvate (PEP) into pyruvate. This reaction also converts ADP and an inorganic phosphate to ATP. This is the third irreversible step of glycolysis and commits PEP to substrate-level phosphorylation. Although it is less regulated than PFK, PK exhibits a special feature: feedforward activation. PK is activated by FBP, the upstream product. By being activated by this intermediate, PK is primed for catalysis. However, like PFK, it can also be inhibited by ATP. This displays how tightly glycolysis is controlled by allosteric regulation alone. However, systemic changes require broader mechanisms that are beyond the scope of allosteric control (Voet and Voet, 2011).
Hormonal Regulation: Covalent Enzymatic Modifications
Hormones play a large role in controlling glycolytic output. Unlike allosteric mediators, they control glycolysis at a systemic level. This is done by releasing hormones from specialized cells and spreading them through the bloodstream. Once signalling pathways have activated by hormone action, the result is often covalent modifications to key metabolic enzymes. Two examples of these hormones are insulin and glucagon.
Insulin Pathway
When blood glucose levels are high, insulin is released to increase uptake. This is done through the insulin receptor: a tyrosine receptor kinase. When insulin binds to its receptor, an autophosphorylation event takes place in the kinase domain. This leads to the activation of a phosphorylation cascade, eventually leading to the activation of phosphatidylinositol-3 kinase (PI3K) and production of PIP3. This starts a pathway that, through Akt signalling, ends with the translocation of GLUT4 channels to the cell membrane. These channels increase the rate of glucose uptake, creating more substrate for glycolysis to act on (Vargas et al., 2022; Koeppen and Stanton, 2017).
In hepatic cells, this mechanism acts in addition to control of PFK-1. The Akt pathway leads to the activation of protein phosphatase 1 (PP1). This enzyme is responsible for the dephosphorylation of several other metabolic enzymes. For example, PP1 dephosphorylates the PFK-2/FBPase bifunctional enzyme. This leads to the PFK-2 subunit being activated, increasing F2,6BP concentrations within the cell. F2,6BP is the most potent allosteric activator of PFK-1, which means it can override the effects of ATP and stimulate glycolysis. A similar case is true for PK, which is dephosphorylated by PP1. This stimulates PK activity (Koeppen and Stanton, 2017).
While insulin activates glycolysis, it must also deactivate any pathways which oppose it. This includes glycogenolysis: the breakdown of glycogen into glucose. Since the role of insulin is to reduce blood glucose levels, this pathway acts against it. To reduce its output, PP1 dephosphorylates glycogen phosphorylase and phosphorylase kinase—two key enzymes involved in glycogenolysis. Through the Akt pathway, insulin also activates phosphodiesterase, which reduces cAMP levels. Since cAMP is a signal for glycogenolysis, this further reduces the flux through that pathway (Koeppen and Stanton, 2017).
Glucagon Pathway
When blood sugar levels are low, glucagon is released. This hormone essentially reverses all the effects of insulin. Glucagon binds to its receptor: a GPCR. This leads to stimulation of the Gs subunit, eventually activating protein kinase A. This kinase phosphorylates several enzymes, reversing the effects of PP1. This is how glucagon and insulin cooperate in an antagonistic manner, shifting metabolic pathways depending on the state of the organism. Sensing this state requires sensing glucose levels, which is dependent on specific gene expression in the pancreas (Koeppen and Stanton, 2017).
Transcriptional Regulation
Tissue-specific gene expression plays a key role in regulating glucose metabolism. The main example of this is HK expression. While cells in the brain and blood express HK-I, cells in skeletal muscle express HK-II. This is significant because HK-II transcription is increased by insulin, committing more glucose to further metabolism. The most unique isoform of HK, however, is HK-IV. Unlike the other three HKs, this isoform does not have a high affinity for glucose and is not inhibited by G6P. Instead, its main role is as a glucose sensor within the liver and β cells of the pancreas. Thus, it is also referred to as glucokinase. When isoforms are expressed in the wrong tissue, several pathological effects can take place. For example, it has been found that cancerous hepatic cells often switch to HK-II instead of glucokinase. This allows faster growth (Perrin-Cocon et al., 2021).
The number of enzymes produced by a cell is also significant to metabolic regulation over longer time spans. As mentioned previously, insulin is responsible for increasing the transcription of HK-II. However, it also lowers transcription of genes for gluconeogenesis. This is done by inactivating the FOXO1 transcription factor through the Akt signalling pathway (Koeppen and Stanton, 2017).
Conclusion
By understanding how glycolysis is regulated within the body, it is easy to see all the ways metabolic pathways can be controlled. Allosteric regulation enables rapid control over the activity of specific enzymes. This is most notable within PFK-1, which is the main commitment point for glycolysis. However, hormones are often better equipped for systemic control. This can be accomplished by the covalent modification—mostly phosphorylation and dephosphorylation—of regulatory enzymes. This is how insulin can control glucose uptake and metabolism across the body. Finally, transcriptional regulation of enzyme expression also plays a big role in how glucose metabolism is regulated. Hepatic cells express glucokinase, a specific isoform of hexokinase which acts as a glucose sensor within the cell. Errors in metabolic regulation often have pathological effects, as seen with HK-II expression in hepatocellular carcinoma. Thus, it is essential to continue research into how glucose metabolism is regulated, as it may give clues as to how many diseases could be treated.
Works Cited
Koeppen, Bruce M, and Bruce A Stanton. Berne and Levy Physiology. 7th ed., Saintt Louis Elsevier Health Sciences, 2017.
Perrin-Cocon, Laure, et al. “A Hexokinase Isoenzyme Switch in Human Liver Cancer Cells Promotes Lipogenesis and Enhances Innate Immunity.” Communications Biology, vol. 4, no. 1, 16 Feb. 2021, https://doi.org/10.1038/s42003-021-01749-3.
Vargas, Elizabeth, and Maria Alicia. “Biochemistry, Insulin Metabolic Effects.” Nih.gov, StatPearls Publishing, 26 Sept. 2022, www.ncbi.nlm.nih.gov/books/NBK525983/.
Voet, Donald, and Judith G Voet. Biochemistry. 4th ed., Hoboken, N.J., John Wiley And Sons, 2011.
Wu, Chaodong, et al. “Regulation of Glycolysis—Role of Insulin.” Experimental Gerontology, vol. 40, no. 11, Nov. 2005, pp. 894–899, https://doi.org/10.1016/j.exger.2005.08.002. Accessed 29 May 2020.