Sarah Robinson, Summer 2020. Partly based on a case by Stephanie Halmo Introduction to Metabolism: End of Module Assignment Honor Statement: This assignment adheres to the standards of UGA’s Culture of Honesty policy. The answers to these questions are my own words and all external sources have been cited. (You do not need to use proper citation formatting, just copy and paste the link.) The observable “flux” of a pathway is the net result of forward and reverse processes. Enzymes control rates of forward and reverse reactions in a pathway, and their activity is highly regulated. Enzyme-mediated regulatory mechanisms allow pathways to be sensitive and responsive to the needs of the organism. 1. Identify the key properties of allosteric proteins and describe the structural basis for these properties. 2. Define anabolism and catabolism. 3. Explain the chemical reasons that ATP is the energy currency in biology, specifically, the negative DG of ATP hydrolysis and the high phosphoryl-transfer potential of ATP. 4. Define flux and predict how the flux of a metabolic pathway changes in response to allosteric regulation. 5. Define the R and T state of an allosteric enzyme and predict the effect of allosteric activators and inhibitors on the R-T state equilibrium. Below is the pathway of Glycolysis: each number circled in yellow is a enzyme-catalyzed step of the pathway. Only the enzymes that catalyze steps 1 (hexokinase), 3 (phosphofructokinase) and 10 (pyruvate kinase) are shown. Introduction to Biochemistry and Molecular Biology End of Module Assignment BCMB 3100E Spring 2022 S Take-Home Point Learning Objectives Glycolytic flux1. The first step of the glycolysis is catalyzed by the enzyme hexokinase. Looking at the image above, the substrates for the enzyme are ____________ and the products are _________. The products of this reaction serve as the substrates for the next reaction, and so on until the last step. The final product of the pathway is pyruvate. Highlight your answer. A. Glucose and ATP ; glucose 6-phosphate and ADP B. Glucose and ADP ; glucose 6-phosphate and ATP C. ADP and ATP ; glucose and glucose 6-phosphate D. Glucose and glucose 6-phosphate ; ADP and ATP 2. Recall that a coupled reaction is when an energetically favorable reaction is used to drive an energetically favorable reaction. In glycolysis, two reactions involve coupling of ATP hydrolysis to substrate phosphorylation. These two reactions are___________. Highlight your answer. A. Steps 1 and 3 B. Steps 7 and 10 C. Steps 1 and 7 D. Steps 3 and 10 Definition of substrate-level phosphorylation: the formation of ATP from ADP in which the phosphoryl donor is a substrate with a higher phosphoryl transfer potential than that of ATP. (from the book’s glossary) ATP is an important energy currency inside most cells. Consider the structures of ATP, ADP, and AMP. 3. Which of these molecules (ATP, ADP or AMP) is most stable because of electrostatic repulsion? How does this help to explain the high phosphoryl-transfer potential of ATP, i.e., why is ATP hydrolysis so energetically favorable? (book section 15.3)AMP is the most stable, because it has the least number of phosphate groups compared to ADP and ATP. This means less electrostatic repulsion between the negative charges of the phosphate groups, making AMP more stable. The process of ATP hydrolysis going from ATP to ADP or AMP is energetically favorable because we're going from a higher energy /less stable molecule in ATP to ADP or AMP, a lower energy/ more stable molecule. This means ATP has a high phosphoryl- transfer potential due to the great amount of free energy liberated in hydrolysis. Of note, in addition to electrostatic repulsion, other reasons why ATP hydrolysis is energetically favorable include: increase in entropy as one molecule of ATP is converted two molecules (i.e. ADP + Pi (orthophosphate) and AMP + PPi (pyrophosphate)), stabilization of ADP and Pi by water molecules and therefore making the reverse reaction less favorable (i.e. ADP to ATP) and greater resonance stabilization of Pi (one of the products of ATP hydrolysis) compared to any of the phosphates in ATP. To understand the regulation of a metabolic pathway, you need to see the “big picture” of the pathway, and not focus on each little step in the pathway. What is the end product of the pathway, its overall goal? Cells are not wasteful and the regulation of pathways makes sense if you reason through it, and don’t overthink too much. 4. Looking at the pathway of Glycolysis shown above, is it a catabolic or anabolic pathway? To decide, examine the steps where ATP is used/made and determine if overall the pathway generates a net amount of ATP or uses a net amount of ATP for each glucose molecule. Highlight your answer. A. anabolic because the pathway generates a net amount of ATP B. anabolic because the pathway uses a net amount of ATP C. catabolic because the pathway generates a net amount of ATP D. catabolic because the pathway uses a net amount of ATP Go to this website: https://biochem.web.utah.edu/iwasa/metabolism/index.html On the website, read the introduction and Chapter 1. The website defines metabolic flux as the “passage of a metabolite through a given pathway over time.” 5. Explain metabolic flux in your own words in 2-3 lines to a friend or family member. Metabolic flux is the interconnected passageway of a metabolite through a reaction system over time regulated by enzymes of the pathway. It works similarly to the flow of traffic in which there are multiple pathways that are faster or slower. Similar to traffic if there is a block in the road, one may take an alternative route, which is the same process in metabolic flux. 6. If a new roadblock appeared at the turn of Warburg St. in the Google Map image, which pathway(s) would be affected, and which metabolites would build up if you compare it to the biochemical pathway in the image to the right? In this scenario, the conversion of pyruvate to lactate conversion would be expected to slow down, resulting in an increase in pyruvate concentration. More pyruvate would shift towards the path where it is converted to AcCoA and oxaloacetate and enter the TCA (tricarboxylic acid) cycle. Read Chapter 2 on the website and watch the videos for Glycolysis and Glycolysis (Zoomed). The conversion of fructose 6-phosphate to fructose 1,6-bisphosphate is the