Answer Key: Cellular Metabolism, Part 1

BIOL230W Week 13 – Cellular Metabolism, Part 1 Many of the processes we’ve discussed in class require energy, typically in the form of GTP or ATP. Where does GTP/ATP come from? How do cells use the uptake of macromolecules (like sugars) to fuel the synthesis of GTP/ATP. Over the next two weeks we will explore how cells transform energy into usable forms. We will use this information to better understand how cancerous cells adapt normal metabolism to fuel uncontrollable cell division. Day 1 Learning Objectives 1. Describe how cells obtain energy sources. 2. Identify the different types of membrane transport proteins based on energetics (passive vs. active transport), direction of transport, and concentration of transported molecules. 3. Analyze how different types of membrane transporters work together to transport glucose transport across the membrane of intestinal epithelial cells. Small Group Activity: How Do Cells Get Energy Brainstorm Brainstrom with your neighbors the following questions: 1. What types of molcules can be used as a source for energy? Carbohydrates (sugars), proteins, and fats 2. How do single-cell organisms obtain energy sources? Do all organisms use the same processes? They will engulf food sources via phagocytosis (heterotrophs). Autotrophs make their own food. 3. How do multicellular organisms obtain energy sources? Do all organisms use the same processes? We eat food since we are heterotrophic and it is absorbed into cells in gut. Autotrophs make the food and transport to rest of body. Mini-Lecture: Membrane Transporters 4. Describe the difference between passive and active transport. ChannelPlease include whether molecules are moving UP or DOWN Facilitated Mediated Passive their concentration gradients. Diffusion Transporter Transport In passive transport, an ion moves across membrane (down gradient, high to low concentration). In active transport, an ion moves across the membrane via carrier protein (up gradient, low to high concentration). 5. The image to the right represents the different types of transport you read about in the Exploration Homework. Label the image with the correct terms:  Active vs. Passive tranport Active  Faciltitate Diffusion Transport  Channel-mediated transporters  Carrier-mediated transporters  Pumps 1 CarrierMediated Transporter Pumps BIOL230W Week 13 – Cellular Metabolism, Part 1 6. What is the main difference between channel- vs. carrier-mediated transport? Channel-mediated transport does not require energy (not binding to transporter protein), while carriermediated does. 7. How can carrier-mediated transport function in both passive and active transport? The transport protein can bind to the solute and can carry it either up or down a concentration gradient. Small Group Activity: Transporters Working Together Many cellular functions will rely on multiple membrane transport proteins working together. In fact, cellular absorption of glucose in human intestines uses three different transporters. Fig 1. – Movement of glucose into an intestinal cell from small intestine lumen (opening) to the blood stream. Yellow cells represent small intestine epithelial cells. Note – epithelial cells of blood vessel and connective tissue cells are not shown. Transport in the small intestine allows for efficient absorption of glucose UP its concentration gradient into the cell, so minimal glucose is wasted (i.e., not absorbed).The following concentration gradients exist in this image: Sodium (Na+) is at a high concentration outside the cell (both small intestine lumen and blood) compared to inside the cell. Glucose may start as a higher concentration outside the cell (small intestine lumen, right after eating), but is quickly transported into the cell, so the majority of time glucose is lower outside the cell. On the other hand, glucose is higher concentration in the cell compared to the blood. Finally, potassium (K+) is lower concentration in the blood compared to inside the cell. 8. Using the caption, draw in concentration gradients for all the molecules. 9. What types of transporters are shown in this figure? (Hint: There are three and they are all different in terms of classification). Pump, active (secondary) 10. Starting with the sodium/potassium pump, what is the final result of this transporter in terms of concentration of sodium in the cell versus blood stream and lumen? It is pumping sodium out, lowering concentration of sodium in cell. 2 BIOL230W Week 13 – Cellular Metabolism, Part 1 11. How is the concentration gradient of sodium being used by another membrane transporter? The transporter is using the concentration gradient to pump glucose into the cell. 12. Considering how the membrane transporters work together, what is the final physiological event occurring? To bring glucose into the cell even when concentrations are low in the cell. Day 2 Learning Objectives 1. Define a redox reaction and why this type of reaction is important to metabolism of glucose. 2. Outline the four major ways that biochemical pathways can be regulated and the impact of changing or altering regulation. 3. Define an allosteric site and analyze data to determine how different conditions may impact enzyme function. 4. Compare and contrast the role of reactions at equilibrium and irreversible reactions in the regulation of glycolysis. 5. Describe the cellular location the different steps of glucose metabolism take place. Mini-Lecture: Flux Introduction 13. Define oxidation vs. reduction. Oxidation is the taking of electrons from something, reduction is the addition of electrons to something. 14. Define catabolism vs. anabolism. Catabolism is conversion of large molecules to small molecules; anabolism is building of large molecules from small molecules. 15. Describe in your own words metabolic flux. The rate that molecules pass through metabolic pathways (both anabolism and catabolism). 16. Outline the four main mechanisms that regulate the flux of molecules through a biochemical pathway. Concentration of products (for steps at equilibrium) – direction reaction moves in is determined by concentration of products and reactants, shift towards equilibrium. Allosteric control of an enzyme – for reversible steps, enzymes are inhibited or promoted. Regulation of enzymes post-translationally – make more enzymes, increase, or decrease activity. Small Group Activity: Data Analysis of Pyruvate Kinase Regulation 3 BIOL230W Week 13 – Cellular Metabolism, Part 1 Pyruvate kinase produces pyruvate in the final step of glycolysis. It can be allosterically regulated by several molecules, as well modified by post-translational phosphorylation and/or acetylation. To the left is the result of an experiment where researchers measured the enzymatic activity (K cat/second) of pyruvate kinase in the absence or presence of fructose 1,6 bisphosphate (FBP). Four different pyruvate kinase variants were tested: wildtype (wtPKM2), S37E and S37D (mutated to mimic phosphorylation), and K433 (mutated to mimic acetylation). 17. Does FBP act as an allosteric regulator? Explain your answer. It binds directly to the enzyme and regulates its function so therefore it can be considered an allosteric regulator (activator). 18. Does phosphorylation alter the enzymatic activity of pyruvate kinase? Hint – compare -FBP activity of wildtype with -FBP activity of mutants that mimic phosphorylation. Yes, it lowers the activity of the enzyme. 19. Does acetylation alter the enzymatic activity of pyruvate kinase (ignore FBP here)? Hint – compare FBP activity of wildtype with -FBP activity of mutants that mimic acetylation. No, it does not regulate enzyme activity. 20. Is the△G of the reaction regulated by pyruvate kinase 0 or negative? Why? Delta G would be negative because the directionality of the reaction can be regulated highly. Small Group Activity: Overview of Glycolysis 4 BIOL230W Week 13 – Cellular Metabolism, Part 1 This figure outlines the steps of glycolysis. Notice that the star indicates reaction between the dashed arrow occur TWICE for each molecule of glucose. Please use the image to answer the following questions. 21. For one molecule of glucose, what is the net ATP directly produced? 2 ATP molecules 22. For one molecule of glucose, what is the net NADH 2 produced? What is the fate of NADH 2? 2 NADH2, which later becomes NAD+ after oxidation. 23. Highlight the steps that have a negative DG. 24. What prevents this pathway from proceeding backwards? Another way of thinking about this: What mechanisms ensure that when glucose levels are high, glycolysis pushes forward to produce pyruvate rather than cycling molecules at each individual step. (Hint – it relates to the step you just highlighted). Based on steps 1 and 3 of glycolysis, a phosphate group is added to a substrate so if glycolysis went backwards it would create ATP instead of glucose.