Regulation of Glycolysis: Short and Long-Term Control Mechanisms

Cells maintain stable ATP levels by regulating the flow of glucose through the glycolytic pathway. Key enzymes catalyzing irreversible reactions in this pathway are positioned as crucial enzymes controlling metabolic reactions. Their activity is regulated by allosteric effectors that bind reversibly and enzyme covalent modifications.

Short-term regulation of glycolysis involves complex interactions among ATP consumption, NADH regeneration, and three key allosteric enzymes (hexokinase, phosphofructokinase, and pyruvate kinase). Regulation of glycolytic rate also relies on transient changes in key metabolite concentrations that can balance ATP production and consumption within the cell.

Long-term regulation of glycolysis involves control by hormones such as glucagon, adrenaline, and insulin.

Phosphofructokinase is The Key Enzyme

Phosphofructokinase-1 (PFK-1) is the least efficient of the three rate-limiting enzymes and thus is a crucial control point in the glycolytic pathway. Glucose-6-phosphate can enter the glycolytic pathway or other pathways such as glycogen synthesis and the pentose phosphate pathway. Cells channel glucose into glycolysis through the irreversible reaction catalyzed by phosphofructokinase PFK-1.

This enzyme exists as a tetramer, featuring not only substrate-binding sites but also sites for binding allosteric activators and inhibitors.

F-1,6-BP, ADP, AMP, etc., serve as allosteric activators, while ATP, citrate, etc., act as allosteric inhibitors:

ATP, besides being a substrate for PFK-1, is also the final product of glycolysis. When cellular ATP synthesis exceeds consumption, ATP binds to the allosteric regulatory site of PFK-1, reducing its affinity for fructose-6-phosphate and thus inhibiting PFK-1 activity. Conversely, when ATP consumption surpasses synthesis, resulting in increased ADP and AMP concentrations, the inhibition caused by ATP is alleviated.

Glycolysis serves not only to provide energy under anaerobic conditions but also supplies carbon skeletons for biosynthesis. The demand for carbon skeletons also influences glycolytic rate. High levels of citrate in the cell signify an abundance of biosynthetic precursors, indicating that glucose does not need to be degraded to provide precursor molecules. Citrate inhibits phosphofructokinase activity by enhancing the inhibitory effect of ATP. Additionally, during starvation, the body mobilizes stored fats for oxidation, generating large amounts of acetyl-CoA, which can condense with oxaloacetate to form citrate. This inhibits the activity of phosphofructokinase-1, reducing glucose breakdown to maintain blood glucose concentration during starvation.

Regulation of glycolysis, gluconeogenesis, and triglyceride biosynthesis by insulin, SREBP-1c, and cellular sulfhydryl redox (Hui et al., 2004).

Role of Fructose 2,6-Diphosphate in Glycolysis

2,6-Bisphosphate fructose (F-2,6-BP) is a potent allosteric activator of phosphofructokinase. In the liver, F-2,6-BP enhances the affinity of fructose kinase for fructose-6-phosphate and reduces the inhibitory effect of ATP.

F-2,6-BP is formed from fructose-6-phosphate by phosphofructokinase-2 (PFK2). PFK2 and phosphofructokinase (PFK) are two distinct enzymes with different catalytic mechanisms. The hydrolysis of F-2,6-BP is catalyzed by a specific enzyme, fructose bisphosphatase 2 (FBPase2), producing fructose-6-phosphate. PFK2 and FBPase2 are dual-function enzymes.

Fructose-6-phosphate accelerates the synthesis of F-2,6-BP and also inhibits the hydrolysis of this compound. High concentrations of fructose-6-phosphate can lead to high concentrations of F-2,6-BP. F-2,6-BP further activates phosphofructokinase, known as feed-forward stimulation.

The activities of PFK2 and FBPase2 are regulated by phosphorylation of a serine residue on the enzyme molecule. When glucose is scarce, glucagon in the bloodstream initiates a cAMP cascade, activating cAMP-dependent protein kinase, leading to phosphorylation of PFK2 and FBPase2. FBPase2 is activated, while PFK2 is inhibited, resulting in reduced F-2,6-BP levels and glycolytic inhibition. When glucose is abundant, insulin activates protein phosphatase activity, causing dephosphorylation and activation of PFK2, leading to an increase in F-2,6-BP levels and promotion of glycolysis. This process represents a synergistic control mechanism.

Regulation of Glycolysis by Hexokinase

Hexokinase catalyzes the entry of glucose into the glycolytic pathway. However, glucose-6-phosphate is not the only intermediate in glycolysis; it can also be converted into glycogen or enter the pentose phosphate pathway for oxidation. 2-Deoxyglucose (2-DG), as a glucose analogue, inhibits hexokinase activity.

Hexokinase II in muscle cells exhibits high affinity for glucose. When glucose concentration saturates hexokinase II, muscle hexokinase is inhibited by its catalytic product, glucose-6-phosphate, acting as an allosteric inhibitor.

In the liver, hexokinase IV, also known as glucokinase, is subject to reversible inhibition by glucokinase regulatory protein. Under conditions of high concentrations of fructose-6-phosphate, this nuclear protein binds to glucokinase and localizes it in the nucleus, segregating glucokinase from other glycolytic enzymes located in the cytoplasm, thus inhibiting glycolysis. When blood glucose levels rise, glucose is transported into liver cells by GLUT2. Due to competition between glucose and fructose-6-phosphate for binding to the glucokinase regulatory protein, high concentrations of glucose cause dissociation of glucokinase from the regulatory protein, translocating glucokinase from the nucleus to the cytoplasm, thereby activating glycolysis.

Glucokinase is not inhibited by the reaction product, glucose-6-phosphate, but by fructose-6-phosphate. Fructose-6-phosphate achieves a balance with glucose-6-phosphate through the action of phosphofructokinase. Therefore, even when hexokinases I-III are completely inhibited due to accumulation of glucose-6-phosphate, glucokinase can still function.

Regulation of Glycolysis by Pyruvate Kinase

High concentrations of ATP, acetyl-CoA, and long-chain fatty acids all inhibit the activity of pyruvate kinase. Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate to ATP and pyruvate.

Pyruvate kinase regulates the efflux of pyruvate. The activation of pyruvate kinase by fructose-1,6-bisphosphate facilitates the smooth progression of intermediates in the glycolytic process to the next step. When energy stores are sufficient, the allosteric inhibition of pyruvate kinase by ATP slows down glycolysis. If blood glucose levels drop, phosphorylation of hepatic pyruvate kinase is triggered, rendering the enzyme inactive, thereby reducing glycolysis and stabilizing blood glucose levels.

The allosteric inhibitory effect of alanine on pyruvate kinase also inhibits glycolysis. Alanine is formed by the acceptance of an amino group by pyruvate. An increase in alanine concentration indicates an excess of pyruvate.

The isoenzyme of hepatic pyruvate kinase is regulated by phosphorylation and dephosphorylation, with its active form being the dephosphorylated form and inactive after phosphorylation. When blood glucose concentration decreases, glucagon release triggers phosphorylation of hepatic pyruvate kinase by cAMP-dependent protein kinase, namely protein kinase A (PKA), rendering it inactive, thereby slowing down the use of glucose as fuel in the liver.

Regulation of Glucose Metabolism by Xylulose 5-Phosphate

In the liver, 5-phospho-fructose 2-kinase (PFK-2)/fructose-2,6-bisphosphatase-2 (FBPase-2) is regulated by 5-phospho-ribosyl-1-pyrophosphate (PRPP) generated from the pentose phosphate pathway, which in turn modulates the glycolytic pathway.

Glucose enters liver cells and is converted to glucose-6-phosphate, entering both glycolysis and the pentose phosphate pathway, leading to an increase in 5-phospho-ribosyl-1-pyrophosphate concentration. 5-phospho-ribosyl-1-pyrophosphate activates protein phosphatase PP2A, which phosphorylates the bifunctional enzyme PFK-2/FBPase-2. Dephosphorylation activates PFK-2 and inhibits FBPase-2, increasing the concentration of 2,6-bisphosphate fructose, thus activating glycolysis.

Factors Affecting the Glycolytic Pathway

Factors affecting the glycolytic pathway encompass a range of variables that influence the activity and regulation of enzymes involved in glycolysis. These factors include:

1. pH: Cellular pH levels play a crucial role in modulating the activity of glycolytic enzymes. Changes in pH can alter the ionization state of amino acid residues within enzyme active sites, affecting enzyme-substrate binding and catalytic efficiency. Generally, glycolytic enzymes exhibit optimal activity within a specific pH range, and deviations from this range can impair enzyme function.
2. Temperature: Temperature serves as another critical factor affecting glycolytic enzyme activity. Enzyme kinetics are temperature-dependent, with higher temperatures typically leading to increased molecular motion and collision frequency, thereby enhancing enzyme activity. However, extreme temperatures can denature enzymes, resulting in decreased activity. Each glycolytic enzyme may have an optimal temperature range for maximal activity.
3. Hormonal Regulation: Several hormones play key roles in regulating glycolysis in response to physiological demands. Glucagon, adrenaline, and insulin are well-known regulators of glycolytic activity. For instance, insulin promotes glycolysis by activating enzymes such as phosphofructokinase-1 (PFK-1) and hexokinase, while glucagon stimulates gluconeogenesis, antagonizing the actions of insulin. Additionally, cortisol, growth hormone, and thyroid hormones can also influence glycolytic activity under specific conditions.
4. Substrate Availability: The availability of substrates such as glucose and fructose-6-phosphate directly impacts glycolytic flux. Changes in substrate concentrations can affect the rate of glycolysis by altering the activity of rate-limiting enzymes such as hexokinase and phosphofructokinase. Substrate availability is tightly regulated to meet cellular energy demands and maintain metabolic homeostasis.
5. Allosteric Regulation: Allosteric regulation plays a crucial role in fine-tuning glycolytic flux. Various metabolites, known as allosteric effectors, can bind to allosteric sites on glycolytic enzymes, modulating their activity. For example, ATP acts as a negative allosteric regulator of phosphofructokinase-1 (PFK-1), inhibiting glycolysis when cellular energy levels are high. Conversely, AMP and ADP can activate PFK-1, stimulating glycolysis during energy-demanding conditions.
6. Covalent Modification: Glycolytic enzymes can undergo covalent modifications such as phosphorylation, acetylation, and glycosylation, which can alter their activity and localization within the cell. Phosphorylation, in particular, serves as a key mechanism for regulating enzyme activity in response to signaling pathways activated by hormones and cellular energy status.

Reference

1. Hui, To Yuen, et al. "Mice lacking thioredoxin-interacting protein provide evidence linking cellular redox state to appropriate response to nutritional signals." Journal of Biological Chemistry 279.23 (2004): 24387-24393.