07 Glycolysis

7 Glycolysis

At the beginning of each chapter, we will use an overview map of metabolism to indicate, which pathways are covered in the chapter.

Fig. 1 Overview of nutrient metabolism. Glycolysis is highlighted.
  • Understand how ATP can be generated by breaking down nutrients.
  • Appreciate the design of the glycolytic pathway.
  • Understand how a metabolic pathway can run despite energetic obstacles.
  • Understand how glycolysis is regulated by supply and demand.
  • Breakdown of nutrients
  • Substrate-level phosphorylation
  • Fermentation
  • Enzyme kinetics
  • Allosteric regulation
  • Metabolic coupling

In Chapter 5 you learned how carbohydrates are digested and absorbed into the bloodstream mostly as glucose, fructose and galactose. Because of its central role for energy generation, we will focus on glucose. In blood plasma, glucose levels are maintained at about 5 mM. After a meal they can rise up to 10 mM, but are restored within 2h to fasting levels by storage of glucose in the form of glycogen in muscle and liver.  We will look at the synthesis of glycogen in Chapter 8. Here we look at the metabolism of glucose, which takes place in every cell of the body to generate energy. Per g of weight, the brain is the biggest user of glucose in our body, followed by the heart. The main pathway to break down glucose in our body is called glycolysis (Fig. 2)

Fig. 2 The reactions of Glycolysis. Substrate level phosphorylation steps are underlined in blue.

These reactions appear quite complex, and we will look only at a couple of them in more detail further below. A simpler scheme emphasises the major events in glycolysis (Fig. 3). Glycolysis is often divided into an energy input phase and an energy generating phase. Overall it is an energy generating pathway producing 2 ATP per molecule of glucose and 2 NADH. Glycolysis is subject to metabolic coupling (see chapter 3 for an explanation of this principle); without recycling ATP and NADH the pathway will come to a halt. Glycolysis should be viewed as a supply and demand pathway; i.e. it supplies ATP and NADH for cellular demands. If there is a large demand for ATP and NADH it will run fast, if there is little demand it will run slowly. 

Fig. 3 Overview of glycolysis, its products and substrates. Use of ATP is shaded pink, production of ATP or NADH is shaded green. Phosphorylation of intermediates is indicated by a P in a circle.

The energy investment phase is required to facilitate the aldolase reaction (Fig. 2 Reaction (4)), which has a large positive free energy under standard conditions. Reactions (1) and (3) have large negative free energy under standard conditions. In a chain of reactions where the product of one reaction is the substrate of the next, we can sum up the free energies of all individual reactions to determine the overall energetics of the pathway. If the sum is negative the pathway will proceed to the endproducts, if the sum is positive formation of substrates is favoured. All reactions are in equilibrium with each other, thus reactions can be “pushed” or “pulled” by adjacent reactions. Essentially the aldolase reaction is “pushed” by the investment of 2 ATP upstream. This is illustrated in the table where the actual free energy of the aldolase reaction in an erythrocyte is 0 kJ/mol. 

Reaction in Glycolysis (Fig. 2) Standard free energy (kJ/mol) Free Energy (kJ/mol) in Erythrocytes
1
-16.7
-33.4
2
+1.7
0 kJ/mol
3
-14.2
-22.2
4
+23.8
0 kJ/mol
5
+7.5
0 kJ/mol
6
+6.3
0 kJ/mol
7
-18.8
0 kJ/mol
8
+4.4
0 kJ/mol
9
+7.5
0 kJ/mol
10
-31.4
-16.7

Hexokinase is the first enzyme of glycolysis and one of its important roles is to trap glucose inside the cell. Due to the high affinity and activity of hexokinase, most cells have very low cytosolic glucose concentrations, because it is immediately converted into glucose-6-phosphate. The exception are glucose-sensing cells, such as hepatocytes and beta-cells in the pancreas, where cytosolic glucose concentrations are proportional to blood glucose concentrations. These cells express glucokinase, which has a low affinity for glucose. Thus, flow through glycolysis is regulated by glucokinase instead of phosphofructokinase (see stop & think at the end of the chapter). 

Fig. 4 The role of Hexokinase and Glucokinase in glycolysis.

Reactions (3) and (4) in Fig. 2 are key reactions of the glycolytic pathway. The phosphofructokinase reaction is the main regulator of the glycolytic pathway (see below). The aldolase reaction splits the furanose ring into two molecules of 3 carbon atoms, which prepares them for the conversion to pyruvate and further into acetyl-CoA, the main entry point for full oxidation of nutrients in the tricarboxylic acid (TCA) cycle. The two resulting 3-carbon compounds are interconvertible, which explains the rearrangement of glucose into fructose prior to the aldolase reaction. The next two steps of glycolysis may look familiar, we have investigated the reactions in chapter 3. They are catalysed by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase (Fig. 5).

Fig. 5 Oxidation and Substrate-level phosphorylation during glycolysis.

The energy yield derived from oxidation of glyceraldehyde-3-phosphate is stored through the reduction of NAD and the phosphoryltransfer to the oxidised aldehyde group becoming a phosphorylated carboxyl-group. This reactive acid-anhydride (a condensation between a carboxylic acid and phosphoric acid) is used in the next step to generate ATP. The process is called substrate-level phosphorylation to indicate that a phosphorylated metabolic intermediate is used to transfer a phosphate group onto ADP forming ATP.  To indicate the high-energy of the phosphate group many textbooks use ~P (Squiggle) instead of the linear bond symbol. The phosphate in position 3 of 1,3-Bis-phosphoglycerate, by contrast, is less reactive. It is a phosphate ester, which cannot be used to generate ATP. However, the molecule will be rearranged to generate opportunity for a second substrate-level phosphorylation, which occurs in reaction (10) of Figure 2. This is the pyruvate kinase reaction, which is shown in Fig. 6. In this case activation of the phosphate group is different. When phosphate forms an ester, it would normally have a low free energy of hydrolysis. However, it can only form the ester when pyruvate is in its enol-form, where -OH is available for ester formation, instead of the preferred keto-tautomer. As you saw in the discussion of metabolic principles, keeping reactions out of equilibrium is a frequently used strategy in biochemical energetics. The phospho-enol cannot tautomerise, but once the phosphate is transferred onto ADP, it will rearrange immediately, because the proton can relocalise. This drives the whole phosphoryl-transfer reaction. 

Fig. 6 Substrate level phosphorylation in the final steps of glycolysis.

The whole reaction has a free energy of -61.9kJ/mol of which -30.5kJ/mol are preserved as ATP. Thus, the overall free energy is -31.4kJ/mol.

The overall balance of glycolysis is:

 Glucose + 2ATP + 2NAD+ + 4ADP + 2Pi –> 2 pyruvate +2ADP +2NADH +2H+ + 4ATP +2H2O

Cancelling out common terms reduces the balance to:

 Glucose + 2NAD+ + 2ADP + 2Pi –> 2 pyruvate +2ADP +2NADH +2H+ + 2ATP +2H2O

Because glycolysis produces NADH and ATP it is subject to metabolic coupling (see chapter 3), it can only run when NADH is converted back to NAD and ATP converted back to ADP by cellular processes. There are two ways to recycle NADH back to NAD. The typical pathway in our body is through respiration inside mitochondria. This requires shuttling of NADH equivalents into mitochondria by reversible oxidation and reduction reactions. In certain cell types, such as erythrocytes (no mitochondria) or if oxygen supply is via blood circulation is insufficient (muscle fibers, cancer cells), fermentation is an alternative pathway. In this case NADH is recycled by using it to reduce pyruvate to lactate (Fig. 7). Please note that fermentation generates much less ATP, because most of the energy is still preserved in pyruvate, which instead of being fully oxidised in the mitochondria is converted to lactate. Thus, fermentation shows a much tighter metabolic coupling than respiration. The flow through glycolysis must be elevated during fermentation to generate sufficient ATP. The two modes of glycolysis illustrate the necessity to recycle NADH back to NAD. This also applies to ATP, but the speed of the whole pathway adapts to the amount of ATP required, because NADH can be recycled by two complementary reactions.

During hard exercise so much pyruvate and NADH is produced that shuttling of NADH into mitochondria is not fast enough. Thus, lactate is produced although oxygen levels in muscle might be sufficient, because lactate dehydrogenase is present in large amounts. The maximal rate of ATP resynthesis from the conversion of glycogen into lactate in human muscle is estimated at 1.5 mmol · kg–1 · s–1 and is reached within 5 s of maximal exercise. In contrast, the maximal rate of ATP resynthesis from the conversion of glycogen into CO2 is estimated at 0.5 mmol · kg–1 · s–1 and, to be reached, it requires more than 1 min of maximal exercise.
Fig. 7 Alternative fates of NADH and glycolysis in the absence and presence of oxygen or mitochondria. In the presence of mitochondria and oxygen NADH is reoxidised within mitochondria, allowing glycolysis to proceed to pyruvate and finally the production of carbon dioxide. In the absence of oxygen or mitochondria, NADH is used to reduce pyruvate to lactate. Lactate is then released by the cell, allowing glycolysis to continue.

Glycolysis is a dynamic pathway. Its rate can increase up to 100-fold in exercising muscle. You already learned about regulation of glycolysis through metabolic coupling, i.e. its speed is regulated by the demand for ATP and NADH, generating glycolysis substrates ADP and NAD. However, regulation of glycolysis is even more intricate through direct regulation of two of its enzymes by metabolic end-products or intermediates. Inspection of the free energies of the 10 enzymatic reactions of glycolysis shows that 3 have a significant negative free energy in vivo, namely hexokinase (HK), phosphofructokinase (PFK) and Pyruvate kinase (PK). Thus, these are the driver reactions for the whole pathway and it is not surprising that regulation focuses on HK, PFK and PK. Hexokinase is negatively regulated by its product glucose-6-phosphate, which is used in several different pathways, such as the Pentose-phosphate pathway and the hexosamine pathway, which are regulated independently. 

Fig. 9 Regulation of glycolysis
The inhibition of PFK and PK by ATP is straightforward to understand. Glycolysis is an energy generating pathway that does not need to run if sufficient ATP is available. High levels of citrate indicate that the TCA cycle has sufficient metabolic influx (see chapter 9), allowing glycolysis to slow down, as well. AMP, by contrast, is not a product of glycolysis, but a sensitive indicator of energy depletion. In most metabolic pathways ATP is hydrolysed to ADP avoiding the formation of AMP. However, during high energy demand cells have a rapid energy recovery mechanism through the enzyme adenylate kinase. It catalyses the reaction: ADP+ADP ↔ ATP+AMP. Thus, high levels of ADP are recycled to replenish some ATP but also forming AMP in the process. Because AMP rises on a low background (Insert in Fig. 9) it is easier to detect and serves as an indicator of energy depletion. All cells have signalling proteins that specifically detect AMP to sense the energy status of the cell. We will discuss these proteins in Chapter 18. Fructose-2,6-bisphosphate (F2,6BP) is an important regulator of glycolysis and gluconeogenesis, regulating both pathways in opposite ways. It is generated in response to hormones, and we will discuss it in more detail in chapter 8 and 16. Fructose-1,6-bisphosphate (F1,6BP) is an intermediate of glycolysis that accumulates to drive the aldolase reaction. High levels of Fructose-1,6-bisphosphate indicate a possible “traffic jam” further downstream and therefore activate pyruvate kinase.
To understand how metabolites can regulate enzymes, we must understand more about these proteins and a mechanism called allosteric regulation.
Fig. 10 Allosteric enzymes are multimeric.

In chapter 5 you learned that enzymes bind tightly to the transition state of the substrate and through interactions between enzyme and substrate a chemical reaction is facilitated, which would hardly occur without the enzyme. The binding of a substrate to an enzyme and its conversion to a product has been analysed mathematically by Leonard Michaelis and Maude Menten. When we formalise an enzymatic reaction we can state:

The changes to substrate, enzyme, enzyme-substrate complex and product can be formulated as rate equations and solved mathematically. If you are interested in the derivation of the equation you can watch this video.

To solve the equation, a couple of simplifications are made that can be easily achieved in laboratory experiments.

  • The enzyme rate will be determined in the presence of substrate and enzyme, but hardly any product. This prevents the reversal of the reaction.
  • Substrate is in excess of enzyme and the substrate concentration will essentially remain unchanged during the experiment.
  • Steady state conditions are maintained, substrate is turned over into product at a steady rate.

Under these conditions the reaction velocity (v) has a hyperbolic relationship to the substrate concentration, described by the Michaelis-Menten equation.

Fig. 11 Parameters of enzyme kinetics, enzymes are shown in blue, substrate as an arrow. The grey boxes in the plot highlight important relations between substrate concentration and reaction velocity. Areas A, B and C correspond to the green panels.

As shown in Fig. 11, the reaction velocity (v) increases with increasing substrate concentration. Eventually, the trend tapers off and a maximal velocity (Vmax) is reached. The change in velocity is not achieved by individual enzymes going faster. The velocity indicates how many enzymes are engaged in substrate turnover. Each individual enzyme has a fixed turn-over rate, which does not change. When the substrate concentration is small, only a small fraction of enzymes actually bind a substrate (arrow) (Panel A, corresponding to area A in the curve). At these low substrate concentrations, the reaction rate increases proportionally to the substrate concentration. Panel C (area C) shows the other extreme. Essentially all enzymes are engaged in substrate turnover. An increase of substrate does not further increase the reaction rate. The Michaelis-Menten equation defines a particular concentration (KM) at which 50% of all enzyme molecules are engaged in catalysis (Panel B and area B). This constant is arguably the most frequently measured enzyme parameter in all of the biochemical literature and is called the Michaelis-Menten constant. The KM tells you quite a bit about an enzyme. If you increase substrate concentration 10 x above the KM you are close to Vmax (please note that Vmax is never reached in a hyperbolic curve). If you go to slightly less than 1/2 of KM you are in a dynamic range of the reaction where velocity responds almost linearly to changes of substrate. Literally thousands of enzymes have been characterised in this way and behave as predicted by Michaelis and Menten. However occasionally the curve looks a bit different, namely sigmoidal (Fig. 12, orange curve). 

In Fig. 9, we looked at the regulation of glycolysis and saw that phosphofructokinase (PFK) was activated by AMP. When analysing the kinetic behaviour of the enzyme researchers found a sigmoidal relationship between substrate concentration and reaction rate (Fig. 12, orange curve). However, when adding AMP to the enzyme the relationship looked like a “normal” Michaelis-Menten curve (green curve). What happened?

The interpretation shown in Fig. 12 was initially developed without structural understanding but was later confirmed.

Fig. 12 Principles of allosteric regulation. Allosteric enzymes exist in two states, low affinity (inactive) and high affinity (active). The two states are in equilibrium with each other (1). Binding of substrate will tie the enzyme into the active state (2). Allosteric enzymes are multimeric, all subunits switch synchronously. Substrate does not bind well to the inactive state (3). As substrate concentration increases, more and more subunits switch to active (4). The switch between conformations can also be facilitate by allosteric activators (5) and inhibitors (6).

The curve shape can be explained by assuming a mix of a more active (green) and a more inactive (orange) enzyme population (Fig. 12 (1)). More precisely, one conformation has a low affinity for the substrate (low activity, orange), the other a higher affinity (higher activity, green). In addition these enzymes are multimers (dimers in Fig. 10 and 12) and a switch of one subunit is accompanied by a switch of the partner subunit. In the absence of substrate almost all enzymes are in the more inactive state (low affinity), but some are in the active state (high affinity). Now we add substrate and enzymes that are in the active state will turn over substrate (2). The low-affinity subunits by contrast do not bind substrate very well (3).  Binding of substrate locks the enzymes into the active state and also activates the second subunit (4). Thus, some more inactive enzymes turn active. As we increase the substrate concentration ever more enzymes turn active and the equilibrium shifts towards all enzymes being active at very high substrate concentration. There is a sensitive range (in Fig. 12 between 0.3 and 2 mM substrate), where the enzyme is very sensitive to even small changes of the substrate concentration. This is caused by the dimer design, where activation of one subunit also activates the second subunit. What happens if we add allosteric activator AMP? AMP stabilises the active conformation and pulls more enzymes into the active state (5). In the graph you can see that at 0.5 mM substrate, the enzyme is about 3 times faster in presence of AMP than in the absence. Eventually both enzymes reach the same Vmax, but in the physiologically relevant range the enzyme activity can be changed dramatically. This now explains how we can regulate glycolysis. The key enzymes are multimeric and can switch between an active and inactive conformation. You also saw that there are allosteric inhibitors of PFK (e.g. ATP). These stabilise the inactive conformation (6). On the enzyme the allosteric binding site (next to the green spheres) is in a different position than the substrate binding site (blue circle in Fig. 13) but binding of the allosteric effector impacts the active site.  The changes between the active and inactive state (often called R and T) look rather subtle (Fig. 13), but they are enough to prevent the enzyme from optimal functioning, because the proximity and orientation of substrate in the active site has to be just right.

Fig. 13 Allosteric and catalytic binding sites of phosphofructokinase.

Because ATP is both a substrate and an allosteric inhibitor of PFK, a plot of ATP concentration vs reaction rate reveals a biphasic shape (Fig. 13, red curve). Initially, ATP acts as a substrate and the reaction rate rises. When ATP levels increase, the allosteric inhibitory effect overcomes its role as a substrate. In the presence of AMP (green curve), the effect is blunted.

Question

An allosteric enzyme has the following kinetic properties: a Vmax of 25U/mg enzyme and a half-saturation (K0.5) at 1.0 mM. The kinetic parameters were then measured again in the presence of an allosteric activator. Which of the following would most likely be the finding of the experiment?

a Vmax = 25U; K0.5 = 2.0 mM
b Vmax = 50U; K0.5 = 5.0 mM
c Vmax = 50U; K0.5 = 10.0 mM
d Vmax = 25U; K0.5 = 0.2 mM
e Vmax = 15U; K0.5 = 2.0 mM

Answer

d

  • Glycolysis converts glucose into pyruvate, generating 2 molecules of ATP in the process.
  • Splitting of fructose requires investment of two ATP to offset the equilibrium of this reaction.
  • Substrate level phosphorylation is an important principle of ATP generation involving the transfer of phosphate from glycolysis intermediates onto ADP.
  • Aerobic glycolysis requires transfer of NADH into mitochondria.
  • Anaerobic glycolysis uses NADH to convert pyruvate into lactate.
  • Glycolysis is autoregulated by the energy state of the cell and is also under hormonal control.

Watch the video below to remind you of the steps of glycolysis.

  1. Fig. 1 By the author using ChemDraw
  2. Fig. 2 Thomas Shafee [CC BY 4.0 (https://creativecommons.org/licenses/by/4.0)], from Wikimedia Commons
  3. Fig. 3 By the author using Powerpoint
  4. Fig. 4 By the author using ChemDraw
  5. Fig. 5 By the author using ChemDraw
  6. Fig. 6 By the author using ChemDraw
  7. Fig. 7 By the author using ChemDraw
  8. Fig. 8 Richard W.M. Jones [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/)], via Wikimedia Commons and Photo by Jakes and Associates shared under CC-BY-NC 4.0 
  9. Fig. 9 Modified by the author from Fig. 2 
  10. Fig.10 By the author using Pymol and including Nevermind2 [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/)], from Wikimedia Commons
  11. Fig. 11 By the author using Powerpoint and Origin
  12. Fig. 12 By the author using Origin
  13. Fig. 13 By the author using Origin and Pymol
  14. Fig. 14 By the author using Origin