1. Glycolysis 1: Glycolysis consists of two stages, an ATP investment stage, and an ATP earnings stage Bioc 460 Spring 2008 - Lecture 25 (Miesfeld) Lactate build-up can limit exercise Metabolism of glucose by yeast under anaerobic conditions leads to the production of ethanol and CO 2

2. Metabolic pathways consist of a series of enzymatic reactions that share reactants and products. Metabolic flux (rate of metabolite interconversion) is highly-regulated by both enzyme activity and substrate availability. Nutrient availability affects metabolic flux. Glycolysis is an ancient pathway that cleaves glucose (C 6 H 12 O 6 ) into two molecules of pyruvate (C 3 H 3 O 3 ). Under aerobic conditions, the pyruvate is completely oxidized by the citrate cycle to generate CO 2, whereas, under anaerobic (lacking O 2 ) conditions, it is either converted to lactate, or to ethanol + CO 2 (fermentation). The glycolytic pathway consists of ten enzymatic steps organized into two stages. In Stage 1, two ATP are invested to “prime the pump,” and in Stage 2, four ATP are produced to give a net ATP yield of 2 ATP/glucose. Three glycolytic enzymes catalyze highly exergonic reactions (  G<<0) which drive metabolic flux through the pathway; these enzymes are regulated by the energy charge in the cell (ATP requirements). The three enzymes are hexokinase, phosphofructokinase 1, and pyruvate kinase. Key Concepts in Glycolysis

3. Glucose metabolism in the liver before breakfast Glucagon signaling in liver cells activates both a catabolic pathway (glycogen degradation) and an anabolic pathway (gluconeogenesis), while at the same time inhibiting the catabolism of glucose by the glycolytic pathway.

4. Glucose metabolism in the liver after breakfast Within an hour of eating a bowl of cereal and drinking a cup of fruit juice, your insulin levels increase due to elevated blood glucose causing activation of the insulin signaling pathway and stimulation of glucose uptake, glycogen synthesis, and an increase in glucose catabolism by the glycolytic pathway.

5. Metabolic pathways are linked sets of enzymatic reactions that share common intermediates Remember that substrate concentration and enzyme activity levels affect flux through metabolic pathways

6. There are six major groups of metabolic pathways in nature Metabolic pathways are highly interdependent and exquisitely controlled by substrate availability and enzyme activity levels. Key to understanding metabolic integration in terms of nutrition, exercise, and disease (e.g., diabetes and obesity) is learning how metabolic flux between pathways is regulated and controlled. 1.Anaerobic and aerobic respiration 2.Photosynthesis and carbon fixation 3.Carbohydrate metabolism 4.Lipid metabolism 5.Amino acid metabolism 6.Nucleotide metabolism

7. Should you memorize this chart? http://www.expasy.org/cgi-bin/show_thumbnails.pl

8. Note that you can download a PDF file of this metabolic map from the study guides page on the website. Hierarchical Nature of Metabolism Four classes of macromolecules (proteins, nucleic acids, carbohydrates, and lipids) Six primary metabolite groups (amino acids, nucleotides, fatty acids, glucose, pyruvate, acetyl CoA) Seven small biomolecules (NH 4 +, CO 2, NADH, FADH 2, O 2, ATP, H 2 O)

9. The six major groups of pathways can be subdivided into those responsible for energy conversion, and those involved in the synthesis and degradation of macromolecules. Moreover, the major groups themselves consist of several metabolic modules, all of which will be examined individually.

10. We will use the “divide and conquer” strategy to study metabolism, starting with energy conversion pathways, and then examining the synthesis and degradation of carbohydrates, lipids, and amino acids. Nucleotide metabolism is taught in other courses. The last two lectures in this course (lectures 40 and 41) cover metabolic regulation using diet, exercise, and diabetes as conceptual themes.

11. Four central questions to keep in mind when you study each metabolic pathway 1. What does the pathway accomplish for the cell? 2. What is the overall net reaction of the pathway? 3. What are the key regulated enzymes in the pathway? 4. What are examples of this pathway in real life?

12. Glycolysis: Let’s Answer the Four Questions 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 Pi → 2 pyruvate + 2NADH + 2H + + 2ATP + 2H 2 O ΔGº’ = -35.5 kJ/mol

13. The Four Metabolic Pathway Questions 3. What are the key regulated enzymes in glycolysis? Hexokinase, Phosphofructokinase 1, Pyruvate kinase 4. What are examples of glycolysis in real life? Glycolysis is the sole source of ATP under anaerobic conditions which can occur in animal muscle tissue during intense exercise. Fermentation also relies on glycolysis which is a process that is used to make alcoholic beverages when yeast cells are provided glucose without oxygen.

14. Where does glycolysis fit into the metabolic map? Glycolysis is a central pathway that takes glucose generated by carbohydrate metabolism and converts it to pyruvate. Under aerobic conditions, the pyruvate is oxidized in the citrate cycle which generates reducing power for redox reactions in the electron transport system that result in ATP production by oxidative phosphorylation.

15. Glycolysis takes place entirely in the cytosol, whereas, pyruvate oxidation occurs in the mitochondrial matrix where ATP is generated. Oxygen is not required for glycolysis in the cytosol (anaerobic) but it is necessary for aerobic respiration in the mitochondrial matrix where the O 2 serves as the terminal electron acceptor.

16. The complete oxidation of glucose to CO 2 and H 2 O is highly favorable and releases a large amount of energy that can be harnessed for ATP synthesis Glucose (C 6 H 12 O 6 ) + 6O 2 → 6CO 2 + 6H 2 O ΔGº’ = -2,840 kJ/mol ΔG = -2,937 kJ/mol ΔGº’ for ATP synthesis = -30.5 kJ/mol ΔG for ATP synthesis = ~-50 kJ/mol Theoretical maximum yield = ~60 ATP/glucose Actual yield = 32 ATP/glucose Why are only 32 ATP generated out of a possible ~60 ATP?

17. Pyruvate can also be converted anaerobically to ethanol and CO 2 by fermentation in some micoroorganisms, or converted to lactate

18. For every mole of glucose entering glycolysis, two moles of glyceraldehyde-3-P (GAP) are metabolized to pyruvate, generating in the process a net 2 ATP and 2 NADH. The NADH is a source of reducing power for the cell. Overview of the Glycolytic Pathway

19. The two stages of glycolysis The ATP investment stage generates the high energy intermediate glyceraldehyde-3-P (GAP) which is then oxidized to produce NADH and 1,3- bisphosphoglycerate. The next four reactions lead to the production of FOUR total ATP because each glucose molecule results in the production of TWO pyruvate. The net yield of ATP in glycolysis is therefore TWO ATP.

20. Investment of 2 ATP Production of 2 Glyceraldehyde-3-P (GAP) The two highly regulated steps are hexokinase and phosphofructokinase 1 (both respond directly or indirectly to energy charge). Stage 1

21. Each molecule of GAP Reducing power is captured in the form of NADH; this is a critical step. Phosphoglycerate kinase and pyruvate kinase catalyze a substrate level phosphorylation reaction yielding 4 ATP (2 net ATP). The two pyruvate molecules are further metabolized. Stage 2

22. The six carbons and six oxygens present in glucose are stoichiometrically conserved by glycolysis in the two molecules of pyruvate that are produced. Hydrogen atoms in glucose are lost as H 2 O molecules and in the reduction of NAD +. No loss of carbons or oxygen in glycolysis

23. Chemical features of the glycolytic reactions Ten enzymatic reactions –primarily bond rearrangements –phosphoryl transfer reactions –isomerizations –an aldol cleavage –an oxidation –a dehydration Ideally, you should know the names of all ten enzymes and the reactants and products. The names describe the metabolite structures, draw them if you like, or visualize them in your head. At the very minimum, you need to know which steps the ATP hydrolysis and synthesis takes place, the net reaction of glycolysis, and the three key enzymes the control glycolytic flux.

24. Free energy changes for the ten glycolytic reactions  Gº’ = -35.5 kJ/mol  G = -72.4 kJ/mol

25. Reaction 1: Phosphorylation of glucose by hexokinase or glucokinase Hexokinase is found in all cells. A related enzyme with same enzymatic activity, glucokinase, is present primarily in liver and pancreatic cells.

26. 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 C-6 carbon of glucose

27. Why does it make sense that hexokinase is feedback inhibited by glucose-6-P when energy charge in the cell is high? Hexokinase is feedback inhibited by glucose-6-P which binds to a regulatory site in the amino terminus of the enzyme

28. Reaction 2: Isomerization of glucose-6-P to fructose-6-P by phosphoglucose isomerase Phosphoglucose isomerase (phosphohexose isomerase) interconverts an aldose (glucose-6-P) and a ketose (fructose-6-P) through a complex reaction mechanism that involves opening and closing of the ring structure.

29. Reaction 3: Phosphorylation of fructose-6-P to fructose-1,6-BP by phosphofructokinase 1 Reaction 3 is the second ATP investment reaction in glycolysis and involves the coupling of an ATP phosphoryl transfer reaction catalyzed by the enzyme phosphofructokinase 1 (PFK-1). This is a key regulated step in the glycolytic pathway because the activity of PFK-1 is controlled by numerous allosteric effectors (positive and negative).

30. Reaction 4: Cleavage of fructose-1,6-BP by aldolase to generate glyceraldehyde-3-P and dihydroxyacetone-P The splitting of fructose-1,6-BP into the triose phosphates glyceraldehyde-3-P and dihydroxyacetone-P is the reaction that puts the lysis in glycolysis (lysis means splitting).

31. Reaction 5: Isomerization of dihydroxyacetone-P to glyceraldehyde-3-P by triose phosphate isomerase Glyceraldehyde-3-P, rather than dihydroxyacetone-P, is the substrate for reaction 6 in the glycolytic pathway, making this isomerization necessary. The original TIM barrel structure

32. STAGE 2: ATP EARNINGS Three key features of the very important stage 2 reactions: 1.Two substrate level phosphorylation reactions catalyzed by the enzymes phosphoglycerate kinase and pyruvate kinase generate a total of 4 ATP/glucose (net yield of 2ATP) in stage 2 of glycolysis. 2.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. 3.Reaction 10 is an irreversible reaction that must be bypassed in gluconeogenesis by two separate enzymatic reactions catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase

33. Reaction 6: Oxidation and phosphorylation of glyceraldehyde-3-P by glyceraldehyde-3-P dehydrogenase to form 1,3-bisphosphoglycerate 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.

34. Reaction 7: Generation of ATP by phosphoglycerate kinase in the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate 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. Remember, this occurs twice for every glucose that entered glycolysis. This is an example of a substrate level [ADP] phosphorylation reaction, i.e., ATP synthesis that is not the result of aerobic respiration or photophosphorylation.

35. Reaction 8: Phosphoryl shift by phosphoglycerate mutase to convert 3-phosphyglycerate to 2-phosphoglycerate The purpose of reaction 8 is to generate a compound, 2-phosphoglycerate, that can be converted to phosphoenolpyruvate in the next reaction, in preparation for a second substrate level phosphorylation to generate ATP.

36. The mechanism of this highly reversible reaction requires a phosphoryl transfer from a phosphorylated histidine residue (His-P) located in the enzyme active site The metabolic intermediate 2,3- BPG can diffuse out of active site before it is converted to 2- phosphoglycerate. Remember that 2,3-BPG is important in the regulation of oxygen binding by hemoglobin.

37. Reaction 9: Dehydration of 2-phosphoglycerate by enolase to form phosphoenolpyruvate (PEP) The standard free energy for this reaction is relatively small (ΔGº’ = +1.7 kJ/mol) but it traps the phosphate group in an unstable enol form, resulting in a dramatic increase in the phosphoryl transfer potential of the triose sugar. Standard free energy change for phosphate hydrolysis in 2-phosphoglycerate is ΔGº’ = -16 kJ/mol, whereas the standard freen energy change for phosphate hydrolysis of phosphoenolpyruvate it is an incredible ΔGº’ = -62 kJ/mol !

38. Reaction 10: Generation of ATP by pyruvate kinase when phosphoenolpyruvate is converted to pyruvate 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’ = +30.5 kJ/mol). Unlike phosphoenolpyruvate, pyruvate is a stable compound in cells that is utilized by many other metabolic pathways.

39. Fill in the names of all glycolytic enzymes and metabolites based only on the chemical structures