Glycolysis and Fermentation PDF

# Glycolysis and Fermentation ## Introduction Glucose is often considered the primary fuel source for cells. Not only can glucose be broken down to release energy, but its carbon skeleton can also be used to form other molecules. In addition, byproducts of glucose oxidation can be used as reducing...

# Glycolysis and Fermentation ## Introduction Glucose is often considered the primary fuel source for cells. Not only can glucose be broken down to release energy, but its carbon skeleton can also be used to form other molecules. In addition, byproducts of glucose oxidation can be used as reducing agents to build other molecules or to protect the cell against oxidative damage. This lesson focuses on the catabolism of glucose monosaccharides and how glucose is broken down into two pyruvate molecules. ## 11.1.01 Glycolysis: Energy-Investment Phase The process of glucose catabolism begins with glycolysis. Unlike some other catabolic pathways, which take place in the mitochondria, glycolysis is a metabolic pathway that occurs completely in the cytosol of cells. Glycolysis converts one molecule of glucose into two molecules of a three-carbon compound known as pyruvate while reducing two NAD+ molecules to NADH and producing two net ATP. The net result of glycolysis is shown in Figure 11.1. **Figure 11.1** The net result of glycolysis is the conversion of one molecule of glucose into two molecules of pyruvate and the production of two molecules of ATP and two molecules of NADH. Glycolysis occurs over a series of 10 enzyme-catalyzed reactions, which can be divided into two phases. Although glycolysis ultimately liberates energy in the form of ATP and the reduced cofactor NADH, this can only occur after some energy is invested during the first phase of glycolysis. This concept describes the first five steps of glycolysis, known collectively as the **energy-investment phase**. ## Step 1: Phosphorylation of Glucose by Hexokinase The first step of glycolysis is the irreversible phosphorylation of glucose to form glucose 6-phosphate. Condensation of glucose and inorganic phosphate is thermodynamically unfavorable. Therefore, this phosphorylation reaction requires ATP as the phosphate donor and is catalyzed by the enzyme hexokinase. The substrates and products of the hexokinase reaction are shown in Figure 11.2. **Figure 11.2** Hexokinase catalyzes the first reaction of glycolysis. The phosphorylation of glucose by ATP (instead of inorganic phosphate) is highly exergonic and is therefore considered biochemically irreversible. Much of the free energy change can be explained by the energetic coupling to ATP hydrolysis. This is the first step in which energy is invested into glycolysis by converting ATP to ADP. The irreversible phosphorylation of glucose serves to "trap" glucose in the cell. Glucose typically enters cells by facilitated diffusion through passive transporters. Phosphorylation prevents passive exit from the cell by diffusion because the glucose transporters cannot transport the phosphorylated substrate. Consequently, hexokinase-mediated phosphorylation of glucose commits glucose to the cell; however, it does not necessarily commit glucose to glycolysis. This is because glucose 6-phosphate can enter pathways besides glycolysis, such as the pentose phosphate pathway (Lesson 11.3) or the glycogen synthesis pathway (Lesson 11.4), as shown in Figure 11.3. **Figure 11.3** Several possible fates of glucose 6-phosphate. * *Gluconeogenesis and glycolysis are not typically active at the same time in a given cell.* The exergonic, irreversible nature of the hexokinase reaction means that hexokinase does experience some regulation (particularly in tissues that can also perform gluconeogenesis, see Lesson 11.2); however, because its products are not committed to glycolysis, it is not the major regulated step of glycolysis. ## Step 2: Isomerization of Glucose 6-Phosphate by Phosphoglucose Isomerase The second step of glycolysis is the conversion of glucose 6-phosphate (an aldohexose) into fructose 6-phosphate (a ketohexose, see Figure 11.4). No atoms are gained or lost during this conversion, so this reaction is therefore considered an isomerization reaction. The enzyme that catalyzes this reaction is called phosphoglucose isomerase. Alternate names for the enzyme include phosphohexose isomerase and glucose-6-phosphate isomerase (GPI). **Figure 11.4** Phosphoglucose isomerase catalyzes the second step of glycolysis. This isomerization reaction has a relatively small $ΔG$ under physiological conditions and is therefore considered a reversible reaction. ## Step 3: Phosphorylation of Fructose 6-Phosphate to Fructose 1,6-Bisphosphate by Phosphofructokinase-1 The third step of glycolysis is another phosphorylation step. In this case, the phosphate group is added to the carbon at position 1 of fructose 6-phosphate, resulting in the molecule fructose 1,6-bisphosphate (F1,6BP). The enzyme that catalyzes this reaction is called phosphofructokinase-1 (PFK-1). This reaction is the second energy-investment step in the energy-investment phase of glycolysis. The PFK-1 reaction is shown in Figure 11.5. **Figure 11.5** Phosphofructokinase-1 catalyzes the third step of glycolysis. Like step 1, the phosphoryl transfer reaction of step 3 is energetically coupled to ATP hydrolysis and is both highly exergonic and irreversible. Unlike step 1, the product of step 3 (ie, fructose 1,6-bisphosphate) is committed to glycolysis. In other words, in most cells the F1,6BP and the metabolites that appear after it have no alternate pathway to enter until they are converted to pyruvate at the end of glycolysis. Because PFK-1 is the enzyme that catalyzes the earliest committed step of glycolysis, it is also the most regulated enzyme of glycolysis. ## Step 4: Cleavage of Fructose 1,6-Bisphosphate by Aldolase The fourth step of glycolysis results in the cleavage of fructose 1,6-bisphosphate. This converts the 6-carbon compound to two 3-carbon compounds: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP or G3P). This reaction, catalyzed by the enzyme aldolase, is shown in Figure 11.6. **Figure 11.6** Aldolase catalyzes the fourth step of glycolysis. Aldolase is an example of a lyase enzyme. Lyases split molecules by catalyzing elimination reactions and leave behind a double bond or a ring. Aldolase splits the linear form of F1,6BP and forms a double bond between carbon 3 and its hydroxyl oxygen, resulting in a new aldose phosphate. ## Step 5: Isomerization of DHAP to GAP by Triose Phosphate Isomerase The fifth step of glycolysis and the final step of the energy-investment phase-is the isomerization of dihydroxyacetone phosphate to glyceraldehyde 3-phosphate. This reaction is shown in Figure 11.7. **Figure 11.7** Triose phosphate isomerase catalyzes the fifth step of glycolysis. Recall that the aldolase reaction produces one molecule each of DHAP and G3P. Triose phosphate isomerase only acts on DHAP, converting it into a second copy of G3P. ## Summary of the Energy-Investment Phase of Glycolysis The energy-investment phase of glycolysis consists of its first five steps and involves the conversion of one molecule of glucose into two molecules of glyceraldehyde 3-phosphate. During this phase, the energy of two ATP molecules is invested into the pathway at steps 1 and 3. These two steps are highly exergonic (and therefore irreversible). Step 3, catalyzed by phosphofructokinase-1, is the major point of regulation for glycolysis because it is the first committed step of glycolysis. The two G3P molecules can then each enter the second phase of glycolysis separately, during which the invested energy pays off as even more energy is released from the fuel to produce ATP and NADH. The energy-investment phase of glycolysis is summarized in Figure 11.8. **Figure 11.8** A summary of the energy-investment phase of glycolysis. ## 11.1.02 Glycolysis: Energy-Payoff Phase The energy-payoff phase comprises the second half of glycolysis. During these five enzyme-catalyzed reactions, the glyceraldehyde 3-phosphate (G3P) molecules produced at the end of the energy-investment phase undergo a series of oxidation and phosphoryl transfer reactions that produce more than enough ATP and NADH to offset the energy invested in the first five steps. This concept discusses each of the reactions of the energy-payoff phase as a single molecule of G3P is metabolized through it. Importantly, two molecules of G3P are produced at the end of the energy-investment phase. Therefore, the yield of each reaction should be multiplied by two to give the yield per glucose molecule metabolized. ## Step 6: Oxidative Phosphorylation of G3P by Glyceraldehyde-3-Phosphate Dehydrogenase The sixth step of glycolysis, and the first step of the energy-payoff phase, is the reversible oxidation of glyceraldehyde 3-phosphate. The oxygen of an inorganic phosphate group (Pi) replaces the aldehyde hydrogen, thereby oxidizing the aldehyde to the acyl phosphate group of the product 1,3-bisphosphoglycerate. The hydrogen and its electrons, meanwhile, are transferred to NAD+ in the form of a hydride ion, reducing the NAD+ to NADH. This reaction is catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and is shown in Figure 11.9. **Figure 11.9** Glyceraldehyde-3-phosphate dehydrogenase catalyzes the sixth step of glycolysis. NADH is the first unit of energy currency produced during glycolysis. Although NADH does not directly replace the ATP spent in the energy-investment phase, the high-energy electrons transferred to NADH can later enter the electron transport chain and contribute to the production of ATP, as discussed in Chapter 12. Note that inorganic phosphate is used to phosphorylate G3P. Because each phosphate group will be used to produce ATP by substrate-level phosphorylation, this allows glycolysis to produce more ATP than was invested in the first phase of the pathway. In other words, the phosphate groups eventually transferred to ATP come from both the ATP used in the investment phase and the inorganic phosphate added by GAPDH. ## Step 7: ATP Production through Substrate-Level Phosphorylation by Phosphoglycerate Kinase The seventh step produces the first molecule of ATP in the pathway. Acyl phosphate hydrolysis is coupled to ATP synthesis through a phosphoryl transfer reaction catalyzed by phosphoglycerate kinase. The resulting products are 3-phosphoglycerate and ATP. This reaction is shown in Figure 11.10. **Figure 11.10** Phosphoglycerate kinase catalyzes the seventh step of glycolysis. Note that the enzyme is named for the reverse reaction—the enzyme-catalyzed transfer of a phosphoryl group from ATP onto 3-phosphoglycerate. Nevertheless, enzymes catalyze both directions of any given reaction (see Concept 4.1.04); therefore, the name "phosphoglycerate kinase" also accurately describes the enzyme that transfers a phosphoryl group from 1,3-bisphosphoglycerate onto ADP. The phosphoglycerate kinase reaction produces one ATP per G3P molecule that enters the energy-payoff phase; therefore, it produces two ATP molecules per glucose that enters glycolysis. Because two ATP were invested in the energy-investment phase, by this point the ATP investment has been fully recouped (ie, the net amount of ATP produced and consumed at this point is zero). All ATP made by future steps contribute to the net gain of energy currency molecules. ## Step 8: Functional Group Movement by Phosphoglycerate Mutase The eighth step of glycolysis is a reversible isomerization reaction. Specifically, the phosphate group on carbon 3 moves to carbon 2, forming 2-phosphoglycerate. This reaction is catalyzed by the enzyme phosphoglycerate mutase, as shown in Figure 11.11. **Figure 11.11** Phosphoglycerate mutase catalyzes the eighth step of glycolysis. ## Step 9: Dehydration of 2-Phosphoglycerate to Form Phosphoenolpyruvate by Enolase The ninth step of glycolysis is a reversible dehydration reaction. The hydroxyl on carbon 3 and a hydrogen on carbon 2 are eliminated, yielding water and the molecule phosphoenolpyruvate. This reaction is catalyzed by the lyase enzyme enolase, as shown in Figure 11.12. **Figure 11.12** Enolase catalyzes the ninth step of glycolysis. Phosphoenolpyruvate is a high-energy enol phosphate. The phosphate group prevents the thermodynamically favorable tautomerization of the enol to the keto form, making transfer of the phosphate group favorable. ## Step 10: ATP Production through Substrate-Level Phosphorylation by Pyruvate Kinase The final step of glycolysis is catalyzed by the enzyme pyruvate kinase, which catalyzes the irreversible transfer of the phosphate from phosphoenolpyruvate (PEP) to ADP. This forms ATP by substrate-level phosphorylation. This reaction is shown in Figure 11.13. **Figure 11.13** Pyruvate kinase catalyzes the final step of glycolysis. Like phosphoglycerate kinase, pyruvate kinase is named for the reverse reaction. Unlike phosphoglycerate kinase, however, the large free energy change ($ΔG$ ≈ -23.0 kJ/mol) means that pyruvate kinase facilitates phosphate transfer in only one direction under physiological conditions. In other words, pyruvate kinase is only active in glycolysis; it is not active in gluconeogenesis. Pyruvate kinase produces one ATP molecule per PEP molecule. Therefore, it produces two ATP molecules per glucose that enters glycolysis. Because the ATP investment was already recouped by step 7 (phosphoglycerate kinase), the two ATP produced by pyruvate kinase represent the net gain of ATP by the end of glycolysis. ## Summary of Glycolysis The energy-payoff phase begins at step 6 of glycolysis and uses the G3P molecules produced at the end of the energy-investment phase (ie, steps 1–5). For each G3P molecule that enters, the energy-payoff phase produces one NADH molecule and two ATP molecules, and converts the carbon skeleton from G3P to pyruvate. Therefore, per glucose molecule, the energy-payoff phase produces two NADH, four ATP, and two pyruvate, as shown in Figure 11.14. **Figure 11.14** A summary of the energy-payoff phase of glycolysis. This phase occurs twice per glucose because two molecules of glyceraldehyde 3-phosphate are produced during the energy-investment phase. However, when considering the net gain of glycolysis as a whole, the ATP consumed during the energy-investment phase must also be taken into account. These two ATP consumed are subtracted from the four ATP produced, resulting in the net production of only two ATP per glucose. The complete net outcome of glycolysis is summarized in Figure 11.15. **Figure 11.15** The net outcome of glycolysis is the production of two pyruvate, two ATP, and two NADH. Of the 10 enzyme-catalyzed reactions that compose glycolysis, three are highly exergonic and therefore irreversible: step 1, step 3, and step 10. Of those three, phosphofructokinase-1 (PFK-1, step 3) catalyzes the earliest committed step of glycolysis and is the most tightly regulated. ## Concept Check 11.1 Three molecules of free glucose are catabolized by glycolysis. 1) How many ATP molecules are produced by the enzymes phosphoglycerate kinase and pyruvate kinase? 2) How many net ATP molecules are produced by the entire glycolytic pathway? ## 11.1.03 Alternate Entries into Glycolysis In addition to glucose, other metabolites can also be processed by the glycolytic pathway. When this occurs, the metabolites can enter or exit at different points of the pathway. In other words, not all enzymes need to be used. Furthermore, additional enzymes are typically needed to prepare for entry into glycolysis. Because they use some, but not all, enzymes of glycolysis, these alternate substrates have yields or regulatory mechanisms that may differ from the classic pathway. The catabolism of nonglucose monosaccharides provides several examples of metabolites with alternate entry points into glycolysis. The details of nonglucose metabolism are not a high priority for memorization for the exam, but they provide a deeper understanding of the interrelatedness of biochemical pathways and the effect on net yield and regulation of alternate entry points. In addition to glucose, fructose and galactose are the main monosaccharides obtained from the diet. Mannose is another important monosaccharide often incorporated into glycoproteins or glycolipids. These monosaccharides have six carbons each (ie, they are hexoses, Figure 11.16). **Figure 11.16** Various hexoses can be processed through the glycolytic pathway. ## Mannose Catabolism Hexokinase, the enzyme that catalyzes the first step of glycolysis, has broad substrate specificity and can also phosphorylate mannose and fructose on carbon 6. The product of mannose phosphorylation by hexokinase is mannose 6-phosphate. In contrast, the second enzyme of glycolysis, phosphoglucose isomerase, does not act on mannose 6-phosphate. Instead, the enzyme phosphomannose isomerase catalyzes the analogous reaction that converts mannose 6-phosphate to a ketose. Despite acting on different starting substrates, phosphoglucose isomerase and phosphomannose isomerase produce the same product: fructose 6-phosphate. This is because glucose 6-phosphate and mannose 6-phosphate are C2 epimers of each other. Isomerization of an aldose to a ketose changes only the stereochemistry of carbons 1 and 2, making both carbons achiral. Carbons 3-6 all have the same stereochemistry in D-glucose, D-mannose, and D-fructose (and their derivatives), as shown in Figure 11.17. **Figure 11.17** Glucose, mannose, and fructose differ only at positions C1 and C2. After conversion to fructose 6-phosphate, mannose catabolism uses the same enzymes as the traditional glycolysis pathway, as shown in Figure 11.18. **Figure 11.18** Mannose catabolism enters the traditional glycolysis pathway after conversion to fructose 6-phosphate. ## Fructose Catabolism As mentioned previously, hexokinase can act on fructose, converting it to fructose 6-phosphate directly. Fructose 6-phosphate can then proceed through glycolysis without the need for an additional isomerization step. However, glucose competes with fructose for the hexokinase enzyme, so processing of fructose by hexokinase happens only in limited amounts. The liver metabolizes the majority of dietary fructose using the enzyme fructokinase. Unlike hexokinase, fructokinase adds a phosphate group to carbon 1, forming fructose 1-phosphate. Fructose 1-phosphate can be acted upon by aldolase, which splits the molecule to form dihydroxyacetone phosphate (DHAP) and an unphosphorylated glyceraldehyde. The enzyme triokinase then phosphorylates glyceraldehyde to glyceraldehyde 3-phosphate (G3P), and both DHAP and G3P enter glycolysis and proceed along the classic pathway (Figure 11.19). **Figure 11.19** Fructose is converted into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, both of which can then proceed down the typical glycolysis pathway into the energy-payoff phase. This fructose catabolism pathway allows fructose to be metabolized into two molecules of G3P, but it bypasses the critical regulatory enzyme phosphofructokinase-1 (PFK-1). Consequently, excessive fructose intake can lead to unregulated hepatic glycolysis and is associated with nonalcoholic fatty liver disease (NAFLD). ## Galactose Catabolism Galactose is the C4 epimer of glucose and one of the monomers that compose the disaccharide lactose, which is found in milk products. Like fructose, galactose has its own kinase, galactokinase, which phosphorylates the sugar on carbon 1. The resultant galactose 1-phosphate then participates in a transfer reaction with the molecule UDP-glucose. This reaction, catalyzed by galactose-1-phosphate uridylyltransferase (GALT), transfers the UDP group from glucose to galactose, producing UDP-galactose and glucose 1-phosphate (Figure 11.20). **Figure 11.20** In galactose catabolism, galactose 1-phosphate receives a UDP group from UDP-glucose, producing UDP-galactose and glucose 1-phosphate. The enzyme phosphoglucomutase moves the glucose phosphate group from position 1 to position 6, and the newly produced glucose 6-phosphate molecule can then enter glycolysis as normal. Meanwhile, the UDP-galactose molecule is used to regenerate the consumed UDP-glucose. The enzyme UDP-galactose 4-epimerase (GALE) accomplishes this by inverting the stereochemistry of carbon 4, as shown in Figure 11.21. **Figure 11.21** UDP-galactose is converted to UDP-glucose by inversion of the stereochemistry of carbon 4. The regenerated UDP-glucose can then react with more galactose 1-phosphate to be catabolized further, or it can be used in other metabolic processes such as glycogenesis (see Lesson 11.4). Galactose catabolism is summarized in Figure 11.22. **Figure 11.22** Galactose catabolism. ## 11.1.04 Fermentation The net reaction of glycolysis is: Glucose + 2 NAD+ + 2 ADP+ 2 P₁ → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP Under aerobic (ie, abundant oxygen) conditions, pyruvate and NADH can proceed with aerobic respiration in the mitochondria (see Chapter 12), which converts the pyruvate to CO2 and the NADH molecules back into NAD+. Cells and tissues that either do not possess mitochondria (eg, red blood cells) or have limited oxygen availability (eg, muscles under vigorous exercise conditions) are unable to perform aerobic respiration. Without an alternative to aerobic respiration, pyruvate and NADH accumulate, and NAD+ levels deplete, eventually preventing glycolysis from continuing. **Fermentation** is an alternate pathway to respiration through which cells can handle pyruvate molecules and regenerate NAD+ from NADH. Mammalian cells in particular use a type of fermentation called lactic acid fermentation. In this process, NADH is converted to NAD+ by the enzyme lactate dehydrogenase. To power this oxidation reaction, the enzyme also reduces the pyruvate molecule to a lactate molecule. Like glycolysis, this reaction occurs completely in the cytosol and therefore is available even to cells without mitochondria. The lactic acid fermentation reaction is shown in Figure 11.23. **Figure 11.23** The lactic acid fermentation reaction, catalyzed by lactate dehydrogenase. The lactic acid fermentation reaction is reversible and can proceed in either direction, depending on the relative concentrations of the reactants and products (ie, the reaction quotient Q). In cells that cannot perform respiration, NADH levels are high and the reaction tends toward reduction of pyruvate to lactate. In tissues that can perform respiration, NADH levels are relatively low because NADH is consumed by respiration pathways, and NAD+ levels are higher. This shifts the equilibrium toward oxidation of lactate back into pyruvate. Multicellular organisms such as mammals can take advantage of this reversibility by exporting lactate from anaerobic cells to be processed by well-oxygenated cells. The well-oxygenated cells can then process lactate back to pyruvate due to the abundance of oxygen (and therefore NAD+) available. This cycle is elaborated further as part of the Cori cycle, discussed in Lesson 11.2. ## Alcoholic Fermentation In addition to lactic acid fermentation, many microbial organisms are capable of another type of fermentation known as alcoholic fermentation. In alcoholic fermentation, pyruvate is first decarboxylated to form carbon dioxide (CO2) and acetaldehyde. Reduction of acetaldehyde is then coupled to the oxidation of NADH, which regenerates NAD+ and produces ethanol. This pathway involves two enzymes: pyruvate decarboxylase catalyzes the first, irreversible step and alcohol dehydrogenase catalyzes the second, reversible step. The alcoholic fermentation pathway is shown in Figure 11.24. **Figure 11.24** Alcoholic fermentation consists of two steps: decarboxylation and oxidation-reduction. Microbial organisms use alcoholic fermentation to regenerate NAD+ if they cannot use respiration to do so. Unlike the lactate produced during lactic acid fermentation, the CO2 and ethanol produced during alcoholic fermentation are both volatile (ie, they easily evaporate into the gas phase); therefore, alcoholic fermentation also allows for elimination of the excess carbons from pyruvate. Humans and other higher vertebrates do not perform alcoholic fermentation because they lack the pyruvate decarboxylase enzyme needed for the first step of the pathway. However, humans do possess the alcohol dehydrogenase enzyme, which operates in the reverse direction (ie, oxidizing ethanol to acetaldehyde and reducing NAD+ to NADH) following ethanol consumption. Acetaldehyde can be further oxidized to acetate, which can then be converted to acetyl coenzyme A. This molecule, often abbreviated as acetyl-CoA, is metabolized by the citric acid cycle (Lesson 12.1). Acetaldehyde is highly toxic and responsible for many of the unpleasant effects of alcohol consumption. Consequently, low doses of inhibitors that prevent conversion of acetaldehyde to acetate are sometimes used to treat alcoholism; the painful effects increase as acetaldehyde builds up, which provides a disincentive for future alcohol consumption.