15.4: Regulation of Glycolysis

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Learning Goals (ChatGPT o3-mini)

1. Identify Major Glycolytic Control Points:

   • Describe the roles of hexokinase, phosphofructokinase-1 (PFK1), and pyruvate kinase as the primary regulatory enzymes in glycolysis.

2. Understand Hormonal Regulation of Glycolysis:

   • Compare the effects of glucagon and insulin on glycolysis in the liver, highlighting that glucagon (under low serum glucose) inhibits glycolysis and promotes gluconeogenesis and glycogenolysis, whereas insulin (under high serum glucose) stimulates glycolysis and glycogen synthesis.

3. Examine Isozyme Diversity in Hexokinase Regulation:

   • Explain how different hexokinase isozymes (HKI, HKII, HKIII, and HKIV/Glucokinase) vary in their kinetic properties, subcellular localization, and regulatory mechanisms, and how this diversity tailors glucose phosphorylation to the specific metabolic needs of different tissues.

4. Link Hexokinase Activity to Metabolic Fates of Glucose 6-Phosphate:

   • Outline the multiple metabolic fates of glucose 6-phosphate (energy production via glycolysis and oxidative phosphorylation, glycogen synthesis, nucleotide biosynthesis via the pentose phosphate pathway, and hexosamine synthesis) and discuss how hexokinase activity influences these pathways.

5. Analyze Allosteric Regulation of PFK1:

   • Discuss the structure and regulatory properties of PFK1, including its allosteric sites for activators (e.g., ADP, AMP, and fructose 2,6-bisphosphate) and inhibitors (e.g., ATP and citrate), and explain how these modulators reflect cellular energy status.

6. Understand Tissue-Specific Composition of PFK1 Isozymes:

   • Describe how the subunit composition of PFK1 tetramers (composed of muscle, liver, and platelet subunits) varies among tissues and affects the enzyme’s kinetic and regulatory properties.

7. Examine the Role of Pyruvate Kinase in Glycolysis:

   • Detail the reaction catalyzed by pyruvate kinase, its regulation by feedforward activation (by fructose 1,6-bisphosphate) and feedback inhibition (by ATP and alanine), and the tissue-specific expression of its isozymes (L, R, M1, and M2).

8. Explore Hormonal Control of Pyruvate Kinase:

   • Explain how glucagon signaling in the liver, via protein kinase A (PKA), leads to the phosphorylation and inhibition of the liver isozyme of pyruvate kinase, thereby redirecting metabolites toward glucose production rather than glycolysis.

9. Evaluate the Fructose Regulatory Bypass:

   • Describe the alternative pathway for fructose metabolism in the liver and kidneys, where fructose is converted to fructose-1-phosphate and then cleaved by Aldolase B, bypassing key regulatory steps (hexokinase and PFK1) and potentially contributing to metabolic dysregulation.

10. Integrate Hormonal and Cellular Energy Signals:

    • Synthesize how local indicators of cellular energy (ATP, AMP) and systemic hormonal signals (insulin, glucagon, epinephrine) coordinate to regulate the glycolytic pathway, ensuring that glucose utilization is appropriately matched to both the energy demands of individual cells and the overall metabolic state of the organism.

These learning goals aim to provide students with a comprehensive understanding of how glycolysis is tightly regulated at multiple levels—through enzyme isozymes, allosteric modulation, and hormonal signaling—to maintain metabolic homeostasis.

There are three major enzymatic control points within the glycolytic pathway. These include hexokinase, phosphofructokinase, and pyruvate kinase reactions. Key drivers for regulating the pathway are energy demand:

• within the cell, as determined by local indicators such as ATP and AMP
• within the organism as a whole, under the influence of hormone signaling pathways.

We will also see that the regulation of the pathway can vary depending on cell type and cellular needs.

Here is a quick review of some key hormonal effects before we proceed. 

Under low serum glucose conditions:

• Glucagon is secreted.  This turns on glucose synthesis and glycogen breakdown and inhibits glycolysis in the liver.  Glucose is then exported into the blood and restores higher glucose concentration.

Under high serum glucose:

• Insulin is secreted, which promotes glucose uptake in the liver and muscle for energy use.  Glycolysis is activated, gluconeogenesis is inhibited, and storage by glycogen synthase is activated.

Hexokinase Regulation

One of the primary mechanisms that control the regulation of the hexokinase step in glycolysis is the presence of different hexokinase enzymes in different cellular types. Essentially, these are proteins encoded by different genes but perform the same function within the cell. They are known as isozymes. Isozymes can have different enzyme kinetics, tissue expression patterns, and post-translational modifications and bind with different allosteric effectors. This affords the body to have differential control over the same processes in different locations within the body. Four important hexokinase isozymes within vertebrates vary in subcellular locations and kinetic parameters. This allows the differential phosphorylation of hexoses depending on local conditions and physiological function. They are designated hexokinases I, II, III, and IV. All of the hexokinases can use multiple hexoses as substrates in addition to glucose. These include mannose, fructose, and 2-deoxyglucose. Hexokinase IV is also called glucokinase and is specific to the liver and pancreas.

Recall that hexokinase enzymes mediate the first step in the glycolytic pathway with the formation of glucose 6-phosphate, as shown in Figure \(\PageIndex{1}\) below.  They also require ATP as a cofactor in the process.  

Figure \(\PageIndex{1}\): Glucose Phosphorylation by Hexokinase Enzymes. Figure modified from YassineMrabet

Recall that glucose-6-phosphate (G6P) has several potential fates within the body, as shown in Figure \(\PageIndex{2}\). It can be an energy source through glycolysis and aerobic respiration pathways. Short bursts of anaerobic respiration can also be sustained in animals that convert pyruvate into lactate. G6P can also be dephosphorylated in the liver and released back into the bloodstream to maintain homeostatic balance. The pancreas uses G6P as a sensor to determine when to secrete insulin and glucagon. The G6P can also serve as a building block for anabolic processes. It can be converted to ribose through the Pentose Phosphate Pathway where it will be used to synthesize nucleotide monomers. It can also form hexosamines used in proteoglycan and glycoprotein formation.

Figure \(\PageIndex{2}\): Cellular Fates of Glucose 6-Phosphate

Now, let’s look at the different isozymes of hexokinase in a little more detail. Hexokinase I (HKI) is widely distributed throughout the body and is the main form expressed in brain tissue and red blood cells, as shown in Figure \(\PageIndex{3}\). In brain cells, this protein is localized to the mitochondria. This colocalization aids in efficiently coupling glycolysis and the Krebs' cycle/oxidative phosphorylation pathways inside the mitochondria. It also ties the activity of HKI with oxidative phosphorylation and energy load, as HKI preferentially uses mitochondrially-derived ATP in its reaction mechanism. HK's association with the mitochondria also has a protective effect on the cell, reducing the potential for programmed cell death or apoptosis. On the other hand, red blood cells (RBCs) are highly differentiated cells with a very short lifespan. They are replaced in humans approximately every two weeks. RBCs are enucleated and do not have mitochondria, and thus, only generate ATP through glycolysis. The HKI protein is free-floating in the cytoplasm of this system. HKI has a low Km, meaning it has a high affinity for glucose and is active at low substrate concentrations. It is also inhibited by the product glucose-6-phosphate (G6P) in negative feedback inhibition. Essentially, you do not want to waste time and energy making more than you need. Low to moderate levels of free inorganic phosphate can overcome this negative feedback inhibition.

Figure \(\PageIndex{3}\): Localization of Hexokinase I. Hexokinase I (HKI) is found widely throughout the body and is the primary hexokinase found in brain tissue and red blood cells. Within the brain tissue, HKI is localized to the mitochondria. Images from Smart Servier Medical Art and Jessica Polka

HKI and HKII are expressed in the skeletal and heart muscles and insulin-sensitive tissues. While it is thought that HKI is providing a predominantly catabolic role for the use of G6P in energy production, HKII may play a more pertinent role in anabolic processes, providing G6P for conversion to G1P and subsequent utilization in Glycogenesis. Both HKI and HKII are localized to the outer membrane of the mitochondria. However, 95% of HKI is associated with mitochondria, and only about 70% of HKII is, with the remaining HKII fractionating with the cytosolic proteins. This could help to explain the heightened role of HKII in anabolic glycogenesis processes within skeletal muscle and why it is not found in brain tissue. HKII is also often overexpressed in tumor cells, which is associated with higher mortality rates. It has also been linked with the processes of metastasis and with the development of drug resistance. Similar to HKI, HKII also has a low Km and is inhibited by G6P, although the presence of inorganic phosphate does not release this inhibition.

Not much is known about HKIII's functions. It may be an inactive gene duplication or remnant. Under basal conditions, it is not expressed to appreciable levels in any major tissues, and studies on its biological activity show that it is inhibited by glucose at physiological concentrations. However, some studies suggest that it may be expressed during cellular stress responses, such as hypoxia, although its function in these responses is not currently understood.

Hexokinase IV or Glucokinase is specifically expressed within the liver and pancreas. HKIV is cytoplasmic and not tethered to the mitochondria. Activity within the pancreas is a sensor for insulin release and in the liver to produce G6P that will fuel glycogen production. HKIV has a higher Km than HKI and HKII, thus it does not work efficiently at low glucose concentrations. However, it is NOT inhibited by the product, G6P. Thus, it will continue to make G6P, even when levels are high. This helps explain the high levels of glycogen stored within the liver but not elsewhere in the body. This also ensures that the sensor system in the pancreas will accurately read blood glucose levels.

The four isozymes of HK share high homology with one another and appear to have arisen from gene duplication events, as shown in Figure \(\PageIndex{4}\). The left-hand panel of Figure 15.5.4 shows the linear protein domains of the different HK isozymes. HKI and HKII contain an N-terminal domain that localizes the protein to the mitochondrial membrane. HKI, II, and III contain two repeating catalytic domains in the N- and C-terminals. However, mutations in the N-terminal domain in HKI and HKIII render them inactive. Both catalytic domains in HKII retain activity. HKIV (Glucokinase) is the most truncated isozyme, only containing the C-terminal catalytic domain. The lower right diagram shows the ribbon diagram of HKIV. Upstream promoter regions of HKIV (not shown in this diagram) also differ, allowing for controlled expression in the liver and pancreas. Feedback inhibition in HKI and HKII occurs through the N-terminal catalytic domain. The upper diagram on the right shows an HKI dimer complex with an ATP analog, glucose, G6P, and Mg2+ ion. HKI dimerizes when concentrations of the inhibitor, G6P, are high enough. Dimerization reduces the biological activity of the enzyme in brain tissue. Dimerization of HKI can be reversed in the presence of low levels of inorganic phosphate.

Figure \(\PageIndex{4}\): Protein Comparison of the Four Major Hexokinases in Vertebrates. The left-hand panel shows the linear protein domains of the hexokinases I - IV. The upper right diagram shows a dimer of HKI complexed with an ATP analog, glucose, glucose 6-phosphate, and Mg²⁺. The lower panel depicts a ribbon diagram of HKIV. Figures modified from Proteopedia and JAG123

HKIV is also a good model for understanding enzyme conformation change during the reaction. HKs change shape by induced fit upon substrate binding. HKIV has a large induced fit motion that closes over the substrates ATP and xylose, as shown in Figure \(\PageIndex{5}\).  The binding sites are shown in blue, substrates in black, and the Mg²⁺ cofactor in yellow (PDBs:2E2N,2E2Q).

Figure \(\PageIndex{5}\):  HKIV Conformational Change During Substrate Binding. The upper diagram shows the enzyme before substrate binding whereas the lower diagram shows the conformational change upon binding with xylose, ATP, and the Mg²⁺ cofactor. Mg²⁺ is shown in yellow and the substrate binding pocket in the enzyme in blue. Xylose and ATP are shown in black. Figure from Thomas Shafee

In summary, the isozyme expression patterns of HKs differentially regulate the fate of glucose within those tissues. Within brain tissue and red blood cells, where only HKI is present, glucose is predominantly used in the glycolytic pathway for energy production. In muscle tissue, the presence of HKII allows for the increased use of glucose to form glycogen. HKIV expression in the pancreas and liver allows for the homeostatic regulation of blood glucose levels and the stockpiling of glucose in glycogen.

Phosphofructokinase-1 Regulation

Recall that phosphofructokinase-1 (PFK1) mediates the third step in the glycolytic pathway with the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate, as shown in Figure \(\PageIndex{6}\).  The PFK1 reaction is the first irreversible reaction of glycolysis. It also represents the committed step within the pathway. The phosphorylation of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F1,6BP) commits the F1,6BP to continue through the glycolytic pathway. It cannot be utilized for any other purpose at that point. F6P, on the other hand, could be converted back into glucose-6-phosphate and used for many different purposes (ie, glycogen synthesis, nucleotide synthesis, or hexosamine synthesis). Because of the committed nature of this step, PFK1 is one of the most important control points in the glycolytic pathway.

Figure \(\PageIndex{6}\):  Phosphofructokinase-1 Activity During Glycolysis. (CC BY 4.0; Henry Jakubowski via LibreTexts)

The PFK1 enzyme is composed of a tetramer that can contain different combinations of three types of subunits: muscle (M), liver (L), and platelet (P). The composition of the PFK1 tetramer differs according to the tissue type in which it is present. For example, mature muscle expresses only the M isozyme; therefore, the muscle PFK1 is composed solely of homotetramers of M4. The liver and kidneys express predominantly the L isoform. In erythrocytes, both M and L subunits randomly form tetramers, including M4, L4, and the three hybrid forms of the enzyme (ML3, M2L2, M3L). As a result, the kinetic and regulatory properties of the various isoenzymes pools depend on subunit composition. Tissue-specific changes in PFK1 activity and isoenzymes content contribute significantly to the diversities of glycolytic and gluconeogenic rates, which have been observed for different tissues.

PFK1 is an allosteric enzyme with a structure similar to that of hemoglobin insofar as it is a dimer of a dimer, as shown in Figure \(\PageIndex{7}\).  One half of each dimer contains the ATP binding site, whereas the other half has the substrate (fructose-6-phosphate or (F6P)) binding site and a separate allosteric binding site that can bind with ADP or AMP. The allosteric binding of ADP or AMP activates all isoforms of PFK1. This indicates a low energy state within the cell and the need for glycolysis and energy generation. Allosteric inhibitors include high levels of ATP and Citrate. Note that ATP is a substrate of this enzyme and has the normal substrate binding site. When there is enough ATP present that it can also bind allosterically to the enzyme, it will act as an inhibitor. Citrate, the first molecule in the Kreb's Cycle (Citric Acid Cycle), can also act as an allosteric inhibitor of PFK-1. At high citrate levels, pyruvate is not needed to generate ATP through oxidative phosphorylation. We will also see that Fructose 2,6-bisphosphate is predominantly a regulator of PFK1 in Liver Cells, where it serves as an activator.

Figure \(\PageIndex{7}\): Structure of Phosphofructokinase-1. The left-hand diagrams show the ribbon and space-filling models from a side view, whereas the right-hand diagrams have been rotated 90 degrees to show the top-down view. The ATP binding sites are in dark blue, and the fructose 6-phosphate binding sites are in red. A separate allosteric binding site for ADP is also shown. Image modified from Mitternacht, S., and Berezovsky, I.N. (2011)

Before we discuss the formation and use of Fructose 2,6-bisphosphate and its role in the regulation of PFK1, let’s review opposing glucose utilization/production pathways within the liver, as shown in Figure \(\PageIndex{8}\). Two opposing pathways within the liver are glycolysis (the breakdown of glucose) and gluconeogenesis (the formation of glucose). It would be unproductive to have both of these pathways operating at the same time. Thus, if one pathway is needed, the other must be turned off. We previously saw this same type of control for glycogenesis (the formation of glycogen) and glycogenolysis (the breakdown of glycogen). When one of these pathways is upregulated, the other must be downregulated.

Figure \(\PageIndex{8}\): Opposing Pathways of Glucose in the Liver. Image modified from Yan, A., et al (2016) In J Biol Sci 12(12):1544-1554 and Servier Medical Art

In the resting state of the liver, when blood glucose levels are in the homeostatic range, gluconeogenesis will be shut off, as it is very expensive to make glucose, and the liver will only invest in making glucose if blood glucose levels fall critically low (ie, extreme exercise or long-term fasting). In the resting state, the liver will use the glycolytic pathways to supply normal levels of ATP energy to maintain housekeeping processes. Glycogenesis and glycogenolysis will be in balance or equilibrium as needed to augment the supply of glucose entering the system from the bloodstream.

If glucose in the blood drops below homeostatic levels, the pancreas releases glucagon and begins this hormone-signaling pathway that causes the liver to release glucose into the bloodstream. Thus, glucagon signaling leads to the downregulation of glycolysis and glycogenesis to shunt glucose pools to the bloodstream. It also leads to an increase in glycogenolysis, or the breakdown of glycogen. During this time, liver cells predominantly generate ATP from lipids rather than carbohydrates. Thus, glycolysis can be inhibited to promote the release of glucose into the bloodstream.

If cellular demand for glucose is high, liver cells will also turn on the gluconeogenesis pathway and make glucose from non-carbohydrate precursors (which it then exports into the bloodstream for delivery to tissues). This is an energy-intensive pathway. The major site of gluconeogenesis is the liver, with a small amount also taking place in the kidney. Little gluconeogenesis occurs in the brain, skeletal muscle, or heart muscle. It just does not make sense to do that! It costs more energy for cells to make glucose than they can get from breaking it down in oxidative phosphorylation. This cost can be dealt with by doing it in the liver and then releasing it into the bloodstream to fuel activities in the brain, heart, and skeletal muscles.

To do this, glucagon stimulates that lovely signaling pathway that you are all now familiar with (reviewed in Figure \(\PageIndex{9}\))! It activates the G-protein-coupled receptor within the liver, which activates the downstream G-protein. Adenylate Cyclase is activated and produces the second messenger, cAMP. cAMP binds with the CREB protein and activates the transcription of proteins involved in gluconeogenesis. The cAMP also binds with Protein Kinase A and upregulates glycogen phosphorylase activity, resulting in glycogen breakdown. It also down-regulates the activity of glycogen synthase to inhibit glycogenesis. In this section, we will also see how PKA activation will down-regulate the activity of the glycolytic pathway.

Figure \(\PageIndex{9}\): Overview of Glucagon Signaling Pathways within the Liver. Image modified from Yan, A., et al (2016) In J Biol Sci 12(12):1544-1554 and Servier Medical Art

Regulation of the glycolytic pathway in response to this signaling occurs through the regulation of the PFK-2/FBPase-2 Enzyme. The activity of this enzyme is controlled through the PKA signaling cascade. This enzyme is responsible for phosphorylating fructose 6-phosphate to the fructose 2,6-bisphosphate form. Note that this bisphosphate form of fructose is DIFFERENT than the bisphosphate form utilized in the glycolytic pathway. The glycolytic pathway requires fructose 1,6-bisphosphate formed from the PFK1 enzyme (or Step 3 of the glycolytic pathway). The PFK-2/FBPase-2 is a separate enzyme not directly involved in the glycolytic pathway. However, we noted previously that fructose 2,6-bisphosphate can be an allosteric activator of the PFK1 enzyme.

The PFK-2/FBPase-2 Enzyme is a dual-purpose enzyme, as shown in Figure \(\PageIndex{10}\). Half of the protein has kinase activity and can phosphorylate fructose-6-phosphate to fructose-2,6-bisphosphate. The other half of the enzyme contains a phosphatase that can hydrolyze the phosphate group from the 2-position and restore fructose-6-phosphate. Note that both activities are not functional at the same time! The phosphorylation state determines the enzyme activity. When the protein is dephosphorylated, the PFK-2 enzyme is active, leading to the production of fructose-2,6-bisphosphate. This molecule can bind with PFK-1 in the glycolytic pathway and increase its activity. If PKA phosphorylates the protein during glucagon signaling, the FBPase is activated, and the kinase activity is inhibited. This leads to the dephosphorylation of fructose at the 2-position and the release of fructose-6-phosphate.

Figure \(\PageIndex{10}\):  Biological Activity of the PFK-2/FBPase-2 Enzyme. The PFK-2/FBPase-2 is a dual functional enzyme capable of phosphorylating the 2'-position of fructose 6-phosphate using the kinase activity. In contrast, the FBPase activity can remove the phosphoryl group from position 2' of fructose 2,6-bisphosphate, yielding fructose 6-phosphate. The PFK-2 component is active when the protein is dephosphorylated while the FBPase remains inactive. However, the FBPase component becomes active following phosphorylation, and the kinase domain is inhibited. Image modified from Kedrosolan and Hyunsuky

As shown in Figure 15.5.10, the PFK-2/FBPase-2 Enzyme is responsible for phosphorylating fructose 6-phosphate to the fructose 2,6 bisphosphate form. Note again that this bisphosphate form of fructose is DIFFERENT than the bisphosphate form utilized in the glycolytic pathway. The glycolytic pathway requires fructose-1,6-bisphosphate formed from the PFK1 enzyme (or Step 3 of the glycolytic pathway). If there is a lot of fructose-6-phosphate around (ie you just drank high fructose corn syrup in your sugary energy drink) it can support both the formation of fructose 1,6-bisphosphate by PFK1 and the production of fructose 2,6-bisphosphate by PFK-2. Fructose 2,6-bisphosphate will bind with PFK1 and increase its activity, converting fructose 6-phosphate into fructose-1,6-bisphosphate, as shown in Figure \(\PageIndex{11}\).

Figure \(\PageIndex{11}\): Fructose 2,6-bisphosphate is a positive allosteric effector of the PFK1 Enzyme. The upper diagram shows a space filling model of PFK1 with the fructose 2,6-bisphosphate binding sites indicated. Mitternacht, S., and Berezovsky, I.N. (2011), ibid.

However, during glucagon signaling, you must shut down this fast-track upregulation of PFK1 by fructose-2,6-bisphosphate and turn down the glycolytic pathway. To do this, Protein Kinase A will phosphorylate the PFK-2/FBPase-2 enzyme and alter its activity. The kinase activity is inhibited, and the phosphorylase activity is turned on. Figure \(\PageIndex{12}\) summarizes this pathway control. In the presence of glucagon, PKA will phosphorylate the PFK-2/FBPase-2 enzyme, causing the kinase activity to be switched off and the phosphatase activity to be switched on. Dephosphorylation of fructose-2,6-bisphosphate recovers fructose-6-phosphate (F6P). F6P, through the reverse isomerase reaction, can be converted back to glucose-6-phosphate (G6P). G6P will then be transported to the rough endoplasmic reticulum, where it will be dephosphorylated, and free glucose can be released back into the blood.

Figure \(\PageIndex{12}\):  Allosteric Regulation of PFK1 in the Liver in Response to Glucagon Signaling. Image from Kedrosolan

The opposite holds for insulin signaling. High insulin concentrations activate protein phosphatase and the dephosphorylation of the PFK-2/FBPase-2 enzyme. In the dephosphorylated state, PFK-2 activity is high, and the FBPase-2 activity is low, stimulating PFK1 and the glycolytic pathway.

Pyruvate Kinase

Recall that pyruvate kinase mediates the final reaction during glycolysis, producing pyruvate and ATP, as shown in Figure \(\PageIndex{13}\). Like PFK1, this enzyme is a key regulatory component within the pathway.

Figure \(\PageIndex{13}\): Review of the Pyruvate Kinase Reaction. Image modified from YassineMrabet

The pyruvate kinase enzyme is active as a tetramer built from the combination of different isozymes expressed in different tissues. There are three major isozymes of pyruvate kinase: the L form that is predominantly found in the liver, the R form that is predominantly found in erythrocytes, the M1 form in muscle and brain, and the M2 form that is expressed in fetal tissue and at some level in most adult tissues. The L and R forms are splice variants that arise from the same gene locus, and the M1 and M2 forms are also splice variants that arise from the same gene locus.

We will focus on some general regulatory mechanisms common to most pyruvate kinase isozymes, starting with the activator, fructose 1,6-bisphosphate (FBP). Because FBP is an earlier product within the same metabolic cascade, the activation of pyruvate kinase enzymes by FBP is known as feedforward stimulation. All the isozymes, except for the M1 form, are stimulated by the binding of FBP to the enzyme. Similarly, all of the pyruvate kinase isozymes are inhibited by the product of the reaction, ATP (or high energy load), and high levels of alanine. Alanine can be converted to pyruvate in one enzymatic step. Thus, pyruvate serves as a metabolic intermediate in the formation of alanine. High levels of alanine indicate a high energy load within the cell (i.e., the cell is full of building blocks to make new macromolecules and does not require more energy). Thus, high levels of alanine serve as a negative regulator of the pyruvate kinase family of enzymes.

The liver isozyme of pyruvate kinase is also regulated through protein phosphorylation, as shown in Figure \(\PageIndex{14}\). Like the PFK-2 activity of the PFK-2/FBPase-2 enzyme, the liver isozyme of Pyruvate Kinase is also downregulated during glucagon signaling. Protein kinase A phosphorylates Pyruvate Kinase, inhibiting its activity and preventing the conversion of phosphoenolpyruvate to pyruvate. The dual regulation of the glycolytic pathway during glucagon signaling helps to ensure that glucose resources will be diverted away from cellular use by the liver and released into the bloodstream to restore homeostatic blood glucose levels.

Figure \(\PageIndex{14}\): Glucagon Signaling in Liver Cells Down Regulates PFK2 Activity and Pyruvate Kinase Activity. Image modified from Yan, A., et al (2016)

Fructose Regulatory Bypass

Other sugars from the diet can also enter into the glycolytic pathway, as shown in Figure \(\PageIndex{15}\). Galactose is converted in a four-step process to glucose-6-phosphate, and mannose can be converted to fructose-6-phosphate. Within most of the body’s tissues, fructose can also be converted into fructose-6-phosphate by hexokinase. However, in the liver and kidneys, there is an alternative route that fructose from the diet can take to enter into the glycolytic pathway. This pathway is concerning because it bypasses two of the major regulatory steps of the glycolytic pathway, the hexokinase step and the PFK1 step. Within the liver and kidneys, fructose can also be converted into fructose-1-phosphate by the enzyme fructokinase. The other isozyme of aldolase, aldolase B, can cleave the fructose-1-phosphate into two three-carbon units, dihydroxyacetone phosphate, and glyceraldehyde. Dihydroxyacetone phosphate can be converted to glyceraldehyde 3-phosphate by Triose Isomerase and then continue into the glycolytic cascade. Glyceraldehyde can be phosphorylated to Glyceraldehyde 3-phosphate by Triokinase. This unregulated system can flood the Kreb Cycle with high levels of pyruvate if high levels of fructose enter the cell (i.e., from high fructose corn syrup, sucrose, and other sweeteners common to the Westernized diet). The excess pyruvate can then be shunted into fatty acid biosynthesis for long-term storage as triglycerides. If the pathway is overutilized by consuming too much sucrose and high fructose corn syrup, this can lead to hypertriglyceridemia (or the heightened increase of body fat). With this, we will end our discussions of glycolysis. In the next section, we will look at the complementary and opposite pathway, gluconeogenesis.

Figure \(\PageIndex{15}\): Alternate Sugar Sources for Glycolysis

Summary

This chapter explores the intricate regulation of the glycolytic pathway, emphasizing its key control points and how both cellular energy status and hormonal signals coordinate to optimize glucose metabolism. Three major enzymatic steps—mediated by hexokinase, phosphofructokinase-1 (PFK1), and pyruvate kinase—are highlighted as the primary control nodes in glycolysis.

Hexokinase Regulation:
The chapter begins with an overview of hexokinase isozymes, which catalyze the first committed step of glycolysis by phosphorylating glucose to form glucose-6-phosphate (G6P). The four hexokinase isozymes (HKI-IV) exhibit distinct kinetic properties, subcellular localizations, and regulatory behaviors tailored to specific tissue functions. For example, HKI in brain tissue is tightly associated with mitochondria for efficient ATP use, while HKIV (glucokinase) in the liver and pancreas acts as a sensor for blood glucose levels due to its higher Km and lack of product inhibition.

Phosphofructokinase-1 (PFK1) Regulation:
As the first irreversible and committed step of glycolysis, PFK1 converts fructose-6-phosphate to fructose-1,6-bisphosphate. The enzyme functions as an allosteric regulator and exists as a tetramer composed of different subunit combinations that vary among tissues. Its activity is modulated by cellular energy indicators: it is activated by ADP, AMP, and fructose 2,6-bisphosphate (an important regulator in liver cells), while high levels of ATP and citrate serve as inhibitors, thereby aligning glycolytic flux with the cell’s energy demands.

Pyruvate Kinase Regulation:
Pyruvate kinase catalyzes the final step of glycolysis, generating pyruvate and ATP. This enzyme is also subject to complex regulation, including feedforward activation by fructose-1,6-bisphosphate and inhibition by ATP and alanine. Its isozymes, which differ between tissues such as liver (L form), erythrocytes (R form), and muscle/brain (M1 and M2 forms), ensure that glycolytic output matches both energy production and biosynthetic requirements. In the liver, pyruvate kinase activity is further downregulated by glucagon-mediated phosphorylation via protein kinase A (PKA).

Hormonal Influence on Glycolysis:
The chapter underscores the role of systemic hormonal signals in modulating glycolysis. Under high serum glucose conditions, insulin promotes glycolysis and glycogen synthesis by enhancing glucose uptake in tissues like liver and muscle. Conversely, during low serum glucose, glucagon triggers a cascade that inhibits glycolysis, promotes gluconeogenesis and glycogenolysis, and ultimately increases blood glucose levels. Epinephrine, primarily affecting skeletal muscle, also activates glycolysis and glycogenolysis to meet immediate energy demands during stress or exercise.

Fructose Regulatory Bypass:
An important additional concept discussed is the fructose regulatory bypass. In the liver and kidneys, dietary fructose can be metabolized via an alternative pathway that bypasses the key regulatory steps catalyzed by hexokinase and PFK1. This unregulated pathway can lead to excessive production of pyruvate and its subsequent shunting into lipogenesis, contributing to metabolic disorders such as hypertriglyceridemia.

Integration and Homeostasis:
Overall, the chapter illustrates how multiple layers of regulation—ranging from tissue-specific enzyme isozymes and allosteric modulation to hormonal signaling—are integrated to finely tune the glycolytic pathway. This integration ensures that glucose metabolism is dynamically adjusted to satisfy both cellular energy requirements and whole-body metabolic homeostasis.

By linking molecular details with systemic regulation, this chapter provides a comprehensive framework for understanding the complexity of glycolysis and its critical role in energy metabolism and disease.