OVERVIEW OF THE GLYCOLYTIC PATHWAY Glycolysis is considered one of the core metabolic pathways in nature for three primary reasons:

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1 Glycolysis 1 Supplemental Reading Key Concepts - Overview of the Glycolytic Pathway Glycolysis generates a small amount of ATP Preview of the ten enzyme-catalyzed reactions of glycolysis - Stage 1: ATP Investment Reaction 1: Hexokinase Reaction 2: Phosphoglucose isomerase Reaction 3: Phosphofructokinase Reaction 4: Aldolase Reaction 5: Triose phosphate isomerase - Stage 2: ATP Earnings Reaction 6: Glyceraldehyde-3-P dehydrogenase Reaction 7: Phosphoglycerate kinase (substrate level phosphorylation) Reaction 8: Phosphoglycerate mutase Reaction 9: Enolase Reaction 10: Pyruvate kinase (substrate level phosphorylation) KEY CONCEPT QUESTION IN GLYCOLYSIS: What are the two substrate level phosphorylation reactions in glycolysis? Biochemical Applications of Glycolysis: Lactose intolerance is caused by reduced levels of the digestive enzyme lactase which is required to hydrolyze the disaccharide sugar lactose to form glucose and galactose. Purified lactase enzyme is commercially available and can be taken as a pill at meal time to reduce symptoms that occur when bacteria of the Lactobacillus family convert lactose to methane and hydrogen gases in the small intestine. OVERVIEW OF THE GLYCOLYTIC PATHWAY Glycolysis is considered one of the core metabolic pathways in nature for three primary reasons: 1) Glycolytic enzymes are highly conserved amongst all living organisms, suggesting it is an ancient pathway. 2) Glycolysis is the primary pathway for ATP generation under anaerobic conditions and in cells lacking mitochondria such as erythrocytes. 3) Metabolites of glycolysis are precursors for a large number of interdependent pathways, including mitochondrial ATP synthesis. The word glycolysis is derived from the Greek glykys, meaning sweet, and lysis which means to split or break, referring to the splitting of one molecule of glucose into two molecules of pyruvate. We will focus on two aspects of glycolysis, 1) the enzymatic reactions that convert glucose to pyruvate, and 2) the role of glycolysis in providing precursors, especially pyruvate, for other metabolic pathways in humans. 1 of 12 pages

2 Let s begin by answering the four questions in our guidebook that pertain to glycolysis: 1. What does glycolysis accomplish for the cell? Generates a small amount of ATP which is critical under anaerobic conditions. Generates pyruvate, a precursor to acetyl CoA, lactate, and ethanol (in yeast). 2. What is the overall net reaction of glycolysis? Glucose + 2NAD + + 2ADP + 2 P i 2 pyruvate + 2NADH + 2H + + 2ATP + 2H 2 O ΔGº = kj/mol 3. What are the key enzymes in glycolysis? Hexokinase commitment step in glycolysis, inhibited by glucose-6-p (product) Phosphofructokinase activated by AMP (low energy charge) and F- 2,6-BP, inhibited by ATP (high energy charge) and citrate (citrate cycle intermediate) Pyruvate kinase - activated by AMP and fructose-1,6-bisphosphate (feed forward), inhibited by ATP and acetyl CoA (excess energy source) Figure What are examples of glycolysis in real life? A deficiency in the hexokinase-related enzyme glucokinase, leads to a rare form of diabetes which is caused by the inability of liver and pancreatic cells to phosphorylate glucose inside cells when blood glucose levels are elevated. Figure 2. We first need to see where glycolysis fits into our metabolic map. As shown in figure 1, glycolysis sits at the top of a set of four interconnected pathways that are responsible for the complete oxidation of glucose to CO 2 and H 2 O by the following reaction: Glucose (C 6 H 12 O 6 ) + 6O 2 6CO 2 + 6H 2 O ΔGº = -2,840 kj/mol ΔG = -2,937 kj/mol Glycolysis generates a small amount of ATP As shown in figure 2, glycolysis takes place entirely in the cytosol, whereas, pyruvate oxidation to CO 2 and H 2 O occurs in the mitochondrial matrix and requires the citrate cycle, electron transport chain and oxidative phosphorylation. Acetyl CoA is the primary metabolite in energy conversion pathways inside the mitochondrial matrix and is also the major breakdown product of fatty acid oxidation. Glycolysis does not require oxygen and therefore, can function under anaerobic conditions, however, complete oxidation of glucose to CO 2 and H 2 O requires oxygen. The combined reactions of glycolysis, the citrate cycle, electron 2 of 12 pages

3 transport chain and oxidative phosphorylation, yield 32 ATP from glucose oxidation. Glycolysis alone yields only 2 ATP out of the total 32 (6%). Therefore, it is the oxidation of pyruvate (and acetyl CoA) in the mitochondria that generates the majority of ATP (94%) for most cells in an organism. Preview of the ten enzyme-catalyzed reactions of glycolysis Figure 3 shows the molecular structures of glucose and pyruvate to illustrate that the six carbons and six oxygens present in glucose are stoichiometrically conserved by glycolysis in the two molecules of pyruvate that are produced. Figure 4. Bioc Figure Dr. Miesfeld 3. Spring 2008 The ten enzymatic reactions of glycolysis are summarized in figure 4 and involve primarily bond rearrangements brought about by enzymes that catalyze phosphoryl transfer reactions, isomerizations, an aldol cleavage, an oxidation, and a dehydration. There is no net loss of carbon or oxygen atoms. Because of the requirement for ATP hydrolysis in the initial reactions, followed by ATP synthesis in the later reactions, glycolysis is divided into two stages, Stage 1: ATP investment (reactions 1-5) and Stage 2: ATP earnings (reactions 6-10). Figure 5 summarizes the glycolytic pathway showing that for every mole of glucose entering glycolysis, two moles of glyceraldehyde-3-p are metabolized to pyruvate, and in the process, generating a net 2 ATP and 2 NADH. The "nodes" in this wiring diagram represent metabolites in the glycolytic pathway.key reaction steps are highlighted along with the corresponding enzymes. 3 of 12 pages

4 One way to understand how a pathway is regulated is to examine free energy changes (ΔGº and ΔG) that take place in each reaction to identify steps that are critical in driving the overall pathway towards product formation. The free energy changes for the ten glycolytic reactions are listed in Figure 6 including both the ΔGº of each reaction which is measured in the laboratory under standard conditions, and the calculated ΔG value using ΔG = ΔGº +RT ln(mass action ratio) based on measured metabolite concentrations in erythrocytes under steady-state conditions. Figure 5. The reactions catalyzed by the enzymes hexokinase, phosphofructokinase and pyruvate kinase have large negative ΔG values and are therefore considered irreversible reactions under physiological conditions. Although several of the glycolytic reactions are shown to have positive ΔG values, it is likely that the mass action ratios for these reactions do not represent actual metabolite concentrations under conditions of high metabolic flux. A good example of this is reaction 5 catalyzed by the enzyme triose phosphate isomerase which has an estimated ΔG of +2.5 kj/mol, but is nevertheless pulled to the right due to the rapid depletion of glyceraldehyde-3-p by the highly favorable reactions in stage 2 of glycolysis. Indeed, the net ΔG value for glycolysis in erythrocytes under steady-state conditions is overwhelmingly negative (-76.5 kj/mol), confirming that the glycolytic pathway is highly favorable. Figure 6. 4 of 12 pages

5 STAGE 1: ATP INVESTMENT Stage 1 of glycolysis includes five enzymatic reactions that accomplish two tasks. First, using ATP as the phosphate donor, they create phosphorylated compounds that are negatively charged and cannot diffuse out of the cell. These phosphorylated metabolites are highly specific substrates for glycolytic enzymes and are the precursors to the high energy compounds 1,3-bisphoglycerate and phosphoenolpyruvate in stage 2 that are used to generate ATP by substrate level phosphorylation. Second, the aldolase reaction in step 4 splits the six carbon fructose-1,6-bp compound into two halves creating glyceraldehyde-3-p and dihydroxyacetone-p, the latter of which is quickly isomerized to form a second molecule of glyceraldehyde-3-p. Reaction 1: Phosphorylation of glucose by hexokinase or glucokinase The first reaction in glycolysis serves to activate glucose for catabolism by attaching a phosphate group to the C6 position to generate glucose-6-p as illustrated in figure 7. This is the first of two ATP investment steps in stage 1 of glycolysis and uses the free energy released from ATP hydrolysis to drive the phosphoryl transfer reaction. Figure 8 shows the pyranose ring structure of glucose and glucose-6-p. Figure 7. Figure 8. Two enzymes catalyze this phosphorylation reaction, hexokinase which is found in all cells, and glucokinase which is present primarily in liver and pancreatic cells. Hexokinase has a broad range of substrate specificities and also phosphorylates mannose and fructose, whereas, glucokinase is highly specific for glucose. Hexokinase activity is inhibited by the product of the reaction, glucose-6-p, which accumulates in cells when flux through the glycolytic pathway is restricted. As described later, glucokinase has a much lower affinity for glucose and is not feedback inhibited by glucose-6-p. These enzymatic properties facilitate the function of glucokinase as a metabolic sensor of high blood glucose levels. Hexokinase binds glucose through an induced fit mechanism that excludes H 2 O from the enzyme active site and brings the phosphoryl group of ATP into close proximity with the C6 carbon of glucose. As shown in figure 9, the molecular structure of yeast hexokinase in the presence and absence of glucose suggests that two domains of the enzyme are like jaws that clamp down on the substrate through a large conformational change Figure 9. 5 of 12 pages

6 Figure 10. Figure 10 illustrates the induced fit mechanism of hexokinase and also shows that glucose-6-p inhibition of hexokinase activity is mediated by binding of glucose- 6-P to an effector site in the N-terminal domain of the protein. Reaction 2: Isomerization of glucose-6-p to fructose-6-p by phosphoglucose isomerase As shown in figure 11, phosphoglucose isomerase (phosphohexose isomerase) interconverts an aldose (glucose-6-p) and a ketose (fructose-6-p) through a multi-step pathway that involves opening and closing of the ring structure. The reaction is readily reversible (ΔGº = 1.7kJ/mol) and controlled by the level of glucose-6-p and fructose-6-p in the cell. Figure 11. Reaction 3: Phosphorylation of fructose-6-p to fructose-1,6-bp by phosphofructokinase Reaction 3 is the second ATP investment reaction in glycolysis and involves the coupling of ATP hydrolysis to a phosphoryl transfer reaction that is catalyzed by the enzyme phosphofructokinase. The reaction is essentially irreversible with a large decrease in standard free energy (ΔGº = -14.2kJ/mol) and serves as major regulatory site in the pathway through allosteric control of phosphofructokinase activity in response to the energy charge of the cell. As shown in figure 12, phosphorylation of fructose-6-p generates fructose-1,6-bisphosphate (fructose-1,6-bp) which will form two different triose phosphates when cleaved in reaction 4. Note that a bisphosphate compound contains two phosphates on different atoms (C1 and C6), whereas, a diphosphate compound such as ADP, contains two phosphates covalently linked to each other. Figure of 12 pages

7 Reaction 4: Cleavage of fructose-1,6-bp into glyceraldehyde-3-p and dihydroxyacetone-p by aldolase The splitting of fructose-1,6-bp Figure 13. into the triose phosphates glyceraldehyde-3-p and dihydroxyacetone-p is the reaction that puts the lysis in glycolysis (lysis means splitting) as shown in figure 13. The enzyme responsible for this cleavage reaction is aldolase (fructose bisphosphate aldolase), which performs the reverse of an aldol condensation in the context of the glycolytic pathway. The aldolase reaction illustrates the important difference between ΔGº and ΔG values. Under standard conditions, aldol condensation is favored since the standard free energy for the cleavage reaction is highly positive (ΔGº = +23.9kJ/mol). Reaction 5: Isomerization of dihydroxyacetone-p to glyceraldehyde-3-p by triose phosphate isomerase The production of dihydroxyacetone-p by aldol cleavage of fructose-1,6-bp in reaction 4 above creates a slight problem because glyceraldehyde-3-p, not dihydroxyacetone-p, is the substrate for reaction 6 in the glycolytic pathway. This dilemma is solved by the enzyme triose phosphate isomerase which converts the ketose dihydroxyacetone-p to the aldose glyceraldehyde-3-p in an isomerization reaction that is similar to reaction 2 (conversion of glucose-6-p to fructose-6-p), albeit in reverse (this time we need an aldose formed from a ketose). Figure 14 shows the reaction catalyzed by triose phosphate isomerase which completes stage 1 of glycolysis, and at the expense of 2ATPs, produces two moles of glyceraldehyde-3-p for every mole of glucose that is phosphorylated in reaction 1. Figure 14. Figure 15. The enzyme triose phosphate isomerase was introduced earlier in the course as an example of an α/β protein structure called a TIM barrel which was first identified in the molecular structure of triose phosphate isomerase, thus the name TIM. As shown in figure 15, the triose substrate sits in the enzyme active site which is formed by the β strands and is located in the center of the protein. 7 of 12 pages

8 STAGE 2: ATP EARNINGS There are three key features of the stage 2 reactions to keep in mind. First, two substrate level phosphorylation reactions catalyzed by the enzymes phosphoglycerate kinase and pyruvate kinase generate a total of 4ATPs (net yield of 2ATP) in stage 2 of glycolysis. Second, an oxidation reaction catalyzed by glyceraldehyde-3-p dehydrogenase generates 2 NADH molecules that can be shuttled into the mitochondria to produce more ATP by oxidative phosphorylation. Third, reaction 10 is an irreversible reaction that must be bypassed in gluconeogenesis by two separate enzymatic reactions catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase. Reaction 6: Oxidation and phosphorylation of glyceraldehyde-3-p to form 1,3-bisphosphoglycerate by glyceraldehyde-3-p dehydrogenase The glyceraldehyde-3-p dehydrogenase reaction is a critical step in glycolysis because it uses the energy released from oxidation of glyceradehyde-3-p to drive a phosphoryl group transfer reaction using inorganic phosphate (P i ) to produce 1,3-bisphosphoglycerate (figure 16). This coupled reaction requires the coenzyme NAD + and includes the formation of an acyl thioester intermediate within the enzyme active site to conserve the oxidation energy. Since NAD + is required for the oxidation step in this reaction, NAD + must be continually replenished within the cytosol to maintain flux through glycolysis. This is accomplished aerobically in the mitochondrial matrix by the electron transport chain, or anaerobically in the cytosol by the enzyme lactate dehydrogenase which converts pyruvate to lactate. Anaerobic fermentation in yeast regenerates NAD + using the enzyme alcohol dehydrogenase which converts pyruvate to CO 2 and ethanol. Figure 16. Figure 17. Importantly, formation of 1,3- bisphosphoglycerate by this coupled oxidation-phosphorylation reaction generates a metabolite with a standard free energy of hydrolysis that is higher than ATP hydrolysis. This difference in free energies is harnessed by the enzyme phosphoglycerate kinase in reaction 7 to drive the synthesis of ATP by a mechanism called substrate level phosphorylation. Figure 17 shows that another glycolytic intermediate, phosphoenolpyruvate, has a standard free energy of phosphate hydrolysis that exceeds both 1,3-bisphosphoglycerate and ATP. 8 of 12 pages

9 Reaction 7: Substrate level phosphorylation to generate ATP in the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate by phosphoglycerate kinase Phosphoglycerate kinase catalyzes the payback reaction in glycolysis because it replaces the 2 ATP that were used in stage 1 to prime the glycolytic pathway. As shown in figure 18, the high phosphoryl transfer energy present in the substrate is used to phosphorylate ADP to form ATP by the mechanism of substrate level phosphorylation, leading to the conversion of 1,3- bisphosphoglycerate to 3-phosphoglycerate. Remember that two moles of 1,3-bisphophoglycerate are formed from every mole of glucose, therefore this reaction occurs twice and generates 2ATP/glucose. Figure 18. The molecular structure of phosphoglycerate kinase is similar to hexokinase in that it has two lobes (jaws) that each bind one of the substrates (ADP-Mg 2+ or 1,3-bisphosphoglycerate) leading to a large conformational change in the enzyme that brings the substrates close together and excludes H 2 O from the active site. The structures of phosphoglycerate kinase in the open and closed conformations are shown in figure 19. Figure 19. The bioenergetics of reaction 7 emphasize two important concepts we have presented in the chapter. First, reaction 6 (glyceraldehyde-3-p dehydrogenase) and reaction 7 (phosphoglycerate kinase) are coupled reactions in that the large change in standard free energy of reaction 7 (ΔGº = kj/mol) pulls the less favorable reaction 6 (ΔGº = +6.3 kj/mol) to the right through the shared intermediate 1,3- bisphosphoglcerate as shown below: (Rxn 6) Glyceraldehyde-3-P + P i + NAD + 1,3-bisphosphoglycerate + NADH + H + ΔGº = +6.3 kj/mol ΔG = -1.3 kj/mol (Rxn 7) 1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP ΔGº = kj/mol ΔG = +0.1 kj/mol (Coupled) Glyceraldehyde-3-P + P i + ADP + NAD + 3-phosphoglycerate + ATP + NADH + H + ΔGº = kj/mol ΔG = -1.2 kj/mol 9 of 12 pages

10 Second, the actual change in free energy for each of these two reactions is very close to zero (ΔG = -1.3 kj/mol, ΔG = +0.1 kj/mol), and therefore both reactions are in fact reversible inside the cell. Again, this difference in ΔGº and ΔG is due to the mass action ratio which takes into account the actual concentrations of substrates and products that exist in the cell. Why is the reversibility of these two reactions important? The answer is that when flux through gluconeogenesis is high, these two glycolytic reactions can be reversed and thus quickly respond to changing conditions in the cell. Reaction 8: Phosphoryl shift in 3-phosphyglycerate to form 2-phosphoglycerte by phosphoglycerate mutase The purpose of reaction 8 is to generate Figure 20. a compound, 2-phosphoglycerate, that can be converted to phosphoenolpyruvate in the next reaction, in preparation for a second substrate level phosphorylation that generates ATP earnings in step 10. The phosphoglycerate mutase reaction is shown in figure 21. Figure 20. The mechanism of this highly reversible reaction is illustrated in figure 21 where it can be seen to require a phosphoryl transfer Figure 21. from a phosphorylated histidine residue (His-P) located in the enzyme active site. In step 1, the substrate 3- phosphoglycerate binds to the enzyme active site and is phosphorylated in the C2 position by a transfer reaction involving the His-P group. This type of substrate interaction with the enzyme is non-covalent and referred to as a substrate enzyme complex. Phosphoryl transfer from the histidine residue to the C2 atom of the 3-phosphoglycerate creates the short-lived intermediate 2,3- bisphosphoglycerate (BPG). In the second step of the reaction, the C3 phosphate is transferred back to the histidine residue of the enzyme to regenerate His-P, leading to the release of 2-phosphoglycerate and binding of a new molecule of 3-phosphoglycerate in the third step. Note that the BPG formed in step 1 can diffuse out of the active site resulting in dephosphorylated enzyme, and you may remember that in 10 of 12 pages

11 red blood cells BPG has an important role in regulating oxygen binding to hemoglobin in red blood cells. When BPG leaves the active site without re-phosphorylating the His group, the enzyme can only be activated when trace amounts of BPG diffuse back into the active site. Reaction 9: Dehydration of 2- phosphoglycerate to form phosphoenolpyruvate by enolase In this penultimate step in glycolysis, a dehydration reaction catalyzed by the enzyme enolase converts 2-phosphoglycerate, a molecule with only moderate phosphoryl transfer potential, to phosphoenolpyruvate (figure 23), which we have already seen has extremely high phosphoryl transfer potential. It is this high phosphoryl transfer potential in phosphoenolpyruvate that is harnessed in the last reaction in glycolysis to form ATP. Figure 23. It is interesting that the change in standard free energy for this reaction is relatively small (ΔGº = +1.7) kj/mol), meaning that the overall metabolic energy available from 2-phosphoglycerate and phosphoenolpyruvate is similar. However, when enolase converts 2-phosphoglycerate to phosphoenolpyruvate, it traps the phosphate group in an unstable enol form, resulting in a dramatic increase in the phosphoryl transfer potential of the triose sugar. The standard free energy change for phosphate hydrolysis in 2-phosphoglycerate is ΔGº = -16 kj/mol, whereas for phosphoenolpyruvate it is an incredible ΔGº = -62 kj/mol. Reaction 10: Substrate level phosphorylation to generate ATP in the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase In this reaction, the high phosphoryl transfer potential of phosphoenolpyruvate is used by the enzyme pyruvate kinase to generate pyruvate, the end product of glycolysis, and 2 ATP are formed for every glucose molecule entering the pathway. This is the second of two substrate level phosphorylation reactions in glycolysis that couples energy released from phosphate hydrolysis (ΔGº = -62 kj/mol) to that of ATP synthesis (ΔGº = kj/mol) as shown in figure 24. Unlike phosphoenolpyruvate, pyruvate is a stable compound in cells that is utilized by many other metabolic pathways as will be described later. Figure of 12 pages

12 ANSWER TO KEY CONCEPT QUESTION IN GLYCOLYSIS: Substrate level phosphorylation reactions generate ATP by capturing the high phosphate transfer potential of the substrate in a coupled reaction with ADP. The enzymes phosphoglycerate kinase and pyruvate kinase catalyze the two substrate level phosphorylation reactions in stage 2 of glycolysis. Since two molecules of glyceraldehyde-3-p are generated for every molecule of glucose metabolized, each of these substrate level phosphorylation reactions yield 2ATP/glucose. Phosphoglycerate kinase (reaction 7) provides the payback of 2ATP invested in stage 1 of glycolysis to generate fructose-1,6-bp, whereas, reaction 10 catalyzed by pyruvate kinase represents the ATP earnings step in the pathway by generating 2 net ATP. 12 of 12 pages