15.4: Regulation of Glycolysis

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Learning Goals 

 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Hexokinase Isozymes and Tissue-Specific Glucose Fate

• Compare the four vertebrate hexokinase isozymes (HKI–IV) with respect to K_m for glucose, product inhibition by glucose-6-phosphate, subcellular localization, tissue distribution, and domain architecture, and explain how these differences tailor the first step of glycolysis to the distinct metabolic priorities of brain, muscle, and liver.
• Explain why glucokinase (HKIV) lacks product inhibition by glucose-6-phosphate and has a higher K_m than HKI–III, and connect these kinetic properties to its roles as a hepatic glucose sensor in the pancreas and as a driver of glycogen synthesis in the liver.

Phosphofructokinase-1: The Committed Step and Its Regulation

• Explain why PFK1 is considered the primary regulatory enzyme of glycolysis — identifying it as the committed, irreversible step — and describe its allosteric regulation by ATP, AMP/ADP, citrate, and fructose-2,6-bisphosphate in terms of how each effector reflects the cell's energy or nutritional status.
• Describe the bifunctional PFK-2/FBPase-2 enzyme, explaining how its opposing kinase and phosphatase activities are controlled by phosphorylation state, how this determines cytoplasmic levels of fructose-2,6-bisphosphate, and how glucagon signaling via PKA shifts the enzyme from its kinase-active to its phosphatase-active form to suppress PFK1 and redirect glucose toward the bloodstream.

Pyruvate Kinase Regulation and Integration of Glycolytic Control

• Describe the regulation of pyruvate kinase isozymes by feedforward activation (fructose-1,6-bisphosphate), product/energy inhibition (ATP and alanine), and — for the liver L isozyme — PKA-mediated phosphorylation during glucagon signaling, and explain the physiological logic of each regulatory mechanism.
• Integrate the regulatory mechanisms at all three glycolytic control points (hexokinase, PFK1, and pyruvate kinase) into a coherent account of how the liver coordinates glycolysis, gluconeogenesis, and glycogen metabolism in response to the opposing hormonal signals of insulin and glucagon.
• Explain the fructose regulatory bypass in the liver and kidneys — including the roles of fructokinase, aldolase B, and triokinase — and describe why this route bypasses the two major regulatory checkpoints of glycolysis, how it can flood the Krebs cycle with pyruvate, and how excess dietary fructose promotes hypertriglyceridemia.

There are three major enzymatic control points within the glycolytic pathway. These include the reactions of hexokinase, phosphofructokinase, and pyruvate kinase. 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 observe that the regulation of the pathway can vary depending on the cell type and its cellular needs.

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

Under low serum glucose conditions:

• Glucagon is secreted.  This activates glucose synthesis and glycogen breakdown, while inhibiting glycolysis in the liver.  Glucose is then exported into the blood and restores a 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 regulating the hexokinase step in glycolysis is the presence of distinct 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 allows the body to exert differential control over the same processes in different locations. Four important hexokinase isozymes in vertebrates differ in subcellular localization 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 hexokinases can use multiple hexoses as substrates, in addition to glucose. These include mannose, fructose, and 2-deoxyglucose. Hexokinase IV, also known as glucokinase, is specifically expressed in 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.  

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 serve as an energy source through both 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 returned to the bloodstream to maintain homeostasis. 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 via the Pentose Phosphate Pathway, which is used to synthesize nucleotide monomers. It can also form hexosamines, which are used in the formation of proteoglycans and glycoproteins.

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 facilitates efficient coupling between glycolysis and the Krebs cycle/oxidative phosphorylation pathways within mitochondria. It also links HKI activity to oxidative phosphorylation and energy load, as HKI preferentially uses mitochondrially derived ATP in its reaction mechanism. HK's association with mitochondria also protects the cell, reducing the risk of programmed cell death (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, indicating a high affinity for glucose and activity at low substrate concentrations. It is also inhibited by the product glucose-6-phosphate (G6P) through negative feedback. 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 skeletal and cardiac muscles, as well as insulin-sensitive tissues. While it is thought that HKI primarily serves a catabolic role in G6P utilization for energy production, HKII may play a more significant 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 explain the heightened role of HKII in anabolic glycogen synthesis in 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 remains unclear.

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 serves as a sensor for insulin release, and in the liver, it produces glucose-6-phosphate (G6P) that fuels 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 pancreas' sensor system accurately reads 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 each contain an N-terminal domain that targets the proteins to the mitochondrial membrane. HKI, II, and III contain two catalytic domains that repeat at the N- and C-termini. 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 the concentration of its inhibitor, G6P, is sufficiently high. Dimerization reduces the enzyme's biological activity 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 the 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 conformational changes 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 is in blue. Xylose and ATP are shown in black. Figure from Thomas Shafee

In summary, the isozyme expression patterns of HKs differentially regulate glucose fate 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 enables increased glucose utilization for glycogen synthesis. HKIV expression in the pancreas and liver enables the homeostatic regulation of blood glucose levels and the storage of glucose as 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 a tetramer that can contain different combinations of three subunit types: 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 the M4 isozyme. The liver and kidneys predominantly express 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 isoenzyme pools depend on subunit composition. Tissue-specific changes in PFK1 activity and isoenzyme content contribute significantly to the diversities in glycolytic and gluconeogenic rates observed across 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) 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 binds to the normal substrate-binding site. When sufficient ATP is present, it can also bind allosterically to the enzyme, acting as an inhibitor. Citrate, the first molecule in the Krebs' 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 predominantly regulates PFK1 in Liver Cells, where it acts 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 simultaneously. 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 within the homeostatic range, gluconeogenesis is shut off because producing glucose is very expensive. The liver will only invest in glucose production if blood glucose levels fall critically low (i.e., during extreme exercise or long-term fasting). In the resting state, the liver uses glycolysis to maintain normal ATP levels, thereby supporting housekeeping processes. Glycogenesis and glycogenolysis will be in balance, or in equilibrium, as needed to augment the supply of glucose entering the system from the bloodstream.

If blood glucose drops below homeostatic levels, the pancreas releases glucagon, initiating a hormonal signaling pathway that prompts the liver to release glucose into the bloodstream. Thus, glucagon signaling downregulates glycolysis and glycogenesis, shunting glucose into the bloodstream. It also increases glycogenolysis, 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 occurring in the kidneys. Little gluconeogenesis occurs in the brain, skeletal muscle, or cardiac muscle. It doesn't make sense to do that! It costs cells more energy to synthesize glucose than they can obtain by breaking it down via oxidative phosphorylation. This cost can be addressed by using the liver to process it and then releasing it into the bloodstream to fuel brain, heart, and skeletal muscle activity.

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 in the liver, which in turn activates the downstream G-protein. Adenylate Cyclase is activated, producing the second messenger cAMP. cAMP binds with the CREB protein and activates the transcription of proteins involved in gluconeogenesis. cAMP also binds to Protein Kinase A and upregulates glycogen phosphorylase activity, resulting in glycogen breakdown. It also downregulates glycogen synthase activity, thereby inhibiting glycogenesis. In this section, we will also examine how PKA activation downregulates glycolytic activity.

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 PFK-2/FBPase-2 Enzyme. The activity of this enzyme is controlled through the PKA signaling cascade. This enzyme catalyzes the phosphorylation of fructose 6-phosphate to form fructose 2,6-bisphosphate. 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, which is formed by the PFK1 enzyme (Step 3 of the glycolytic pathway). The PFK-2/FBPase-2 is a separate enzyme not directly involved in the glycolytic pathway. However, we previously noted that fructose-2,6-bisphosphate can be an allosteric activator of PFK1.

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, thereby restoring fructose-6-phosphate. Note that both activities are not functional at the same time! The phosphorylation state determines the enzyme's 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, releasing fructose-6-phosphate.

Figure \(\PageIndex{10}\):  Biological Activity of the PFK-2/FBPase-2 Enzyme. PFK-2/FBPase-2 is a dual-functional enzyme capable of phosphorylating the 2'-position of fructose 6-phosphate via its 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, which is produced by the PFK1 enzyme (Step 3 of glycolysis). 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 achieve this, Protein Kinase A phosphorylates the PFK-2/FBPase-2 enzyme, thereby altering 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 phosphorylates the PFK-2/FBPase-2 enzyme, inhibiting its kinase activity and activating its phosphatase activity. Dephosphorylation of fructose-2,6-bisphosphate recovers fructose-6-phosphate (F6P). F6P can be converted back to glucose-6-phosphate (G6P) through reversing the isomerase reaction. 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, leading to 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 composed of different isozymes expressed in various 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 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 pyruvate kinase isozymes are inhibited by the reaction product, ATP (or a high-energy load), and by 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 for making new macromolecules and does not require additional 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 ensure that glucose resources are diverted away from cellular use in the liver and released into the bloodstream to restore homeostatic blood glucose levels.

Figure \(\PageIndex{14}\): Glucagon Signaling in Liver Cells Downregulates 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 the glycolytic pathway, as shown in Figure \(\PageIndex{15}\). Galactose is converted into glucose-6-phosphate through a four-step process, and mannose can be converted into 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 by which dietary fructose can enter the glycolytic pathway. This pathway is concerning because it bypasses two of the major regulatory steps in the glycolytic pathway: the hexokinase and PFK1 steps. Within the liver and kidneys, fructose can also be converted into fructose-1-phosphate by the enzyme fructokinase. The other aldolase isozyme, aldolase B, can cleave fructose-1-phosphate into two three-carbon units: dihydroxyacetone phosphate and glyceraldehyde. Dihydroxyacetone phosphate can be converted to glyceraldehyde 3-phosphate by Triose Isomerase, which then continues into the glycolytic cascade. Glyceraldehyde can be phosphorylated to Glyceraldehyde 3-phosphate by Triokinase. This unregulated system can flood the Krebs Cycle with excess pyruvate if fructose enters the cell in high amounts (e.g., 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 (an elevated level of triglycerides in the blood). With this, we conclude our discussion 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

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter examines the regulatory mechanisms governing the three major enzymatic control points of glycolysis — hexokinase, phosphofructokinase-1, and pyruvate kinase — integrating local energy sensing with hormonal signaling to explain how glycolytic flux is matched to the metabolic needs of different tissues.

The first control point, hexokinase, is primarily regulated by differential isozyme expression. The four vertebrate hexokinases share high sequence homology and appear to have arisen by gene duplication, but differ critically in kinetic parameters, subcellular localization, inhibition profiles, and domain structure. HKI, the predominant form in brain and erythrocytes, has a low K_m for glucose and is inhibited by its product glucose-6-phosphate (G6P) through its N-terminal catalytic domain — a negative feedback mechanism that prevents wasteful ATP consumption when G6P accumulates. Low inorganic phosphate can relieve this inhibition. In the brain, HKI is anchored to the mitochondrial outer membrane, coupling glycolysis tightly to oxidative phosphorylation. In erythrocytes, which lack mitochondria entirely, HKI operates in the cytoplasm and is the sole source of glycolytic initiation. HKII is expressed in muscle and insulin-sensitive tissues, is also mitochondria-associated, and is similarly inhibited by G6P; its partial cytoplasmic localization may facilitate its role in supplying G6P for glycogenesis. HKII overexpression is a hallmark of tumor cells and is associated with metastasis and drug resistance. HKIII has low basal expression and is inhibited by physiological glucose concentrations. Glucokinase (HKIV), expressed exclusively in liver and pancreatic β cells, has a higher K_m than HKI–III, is not inhibited by G6P, and undergoes a pronounced induced-fit conformational change upon substrate binding. These properties make it ideal as a glucose sensor — its activity is proportional to glucose concentration across the physiological range — and explain why the liver continues accumulating G6P (and ultimately glycogen) even at high glucose concentrations.

The second and most important control point is PFK1, which catalyzes the first irreversible, committed step of glycolysis — the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. PFK1 is an allosteric tetramer (with tissue-specific M, L, and P subunits) structurally analogous to hemoglobin in its T-to-R state transitions. Its activity is activated by AMP and ADP (signaling low energy charge), and inhibited by ATP (signaling high energy charge) and citrate (signaling that the TCA cycle intermediates are already abundant). In the liver, fructose-2,6-bisphosphate is a potent allosteric activator of PFK1, produced by the kinase activity of the bifunctional PFK-2/FBPase-2 enzyme when it is dephosphorylated. During insulin signaling, protein phosphatase dephosphorylates PFK-2/FBPase-2, promoting its kinase activity, elevating fructose-2,6-bisphosphate, and stimulating glycolysis. During glucagon signaling, PKA phosphorylates PFK-2/FBPase-2, switching it to the phosphatase-active/kinase-inactive form, depleting fructose-2,6-bisphosphate, and releasing the allosteric activation of PFK1 — thereby suppressing glycolysis and redirecting fructose-6-phosphate toward gluconeogenesis and glucose export.

The third control point is pyruvate kinase (PK), which catalyzes the final ATP-generating step of glycolysis. Multiple tissue-specific isozymes exist: the L form (liver), R form (erythrocytes), M1 form (muscle and brain), and M2 form (fetal and most adult tissues). All isozymes except M1 are subject to feedforward activation by fructose-1,6-bisphosphate, an earlier glycolytic intermediate — a mechanism that ensures the rate of the final step keeps pace with the rate of the committed step. All isozymes are inhibited by high ATP and by alanine (a transamination product of pyruvate that signals amino acid and energy abundance). The liver L isozyme is additionally regulated by PKA-mediated phosphorylation during glucagon signaling, which inactivates it and prevents pyruvate production — complementing the inhibition of PFK1 and ensuring that phosphoenolpyruvate is available for gluconeogenesis rather than being consumed by pyruvate kinase.

The chapter concludes with the fructose regulatory bypass, in which dietary fructose enters the glycolytic pathway in the liver and kidneys via a route that circumvents both the hexokinase and PFK1 regulatory checkpoints. Fructokinase phosphorylates fructose at C1 to yield fructose-1-phosphate, which is then cleaved by aldolase B into dihydroxyacetone phosphate and glyceraldehyde. These three-carbon units are converted to glyceraldehyde-3-phosphate and enter glycolysis downstream of both regulatory control points. Because the unregulated entry of fructose bypasses the cell's normal energy-sensing mechanisms, high dietary fructose from sucrose or high-fructose corn syrup floods the lower half of glycolysis with substrate, driving excess pyruvate into fatty acid synthesis and ultimately causing hypertriglyceridemia — a metabolic consequence of significant public health relevance.