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CHM376 Biochemistry I Fall 2023 Problem Set #8: Protein Function and Oxygen Transport Note : throughout the PowerPoint you will see K a and K A ; K d and K D . Note that the capitalization of the subscript is a matter of style and does not change the meaning of these terms. However, if the K is not capitalized, it carries a different meaning. K a is the same as K A but different from k a or k A . We will introduce the use of the lowercase k in the following lecture. Also note that K a (or K A ) used here is distinct from the K a (or K A ) of acid base chemistry. Although they are both equilibrium constants, they describe different phenomena. 1. What is a ligand? What is a receptor? 2. Can a ligand bind anywhere on a receptor or is there a specific part of the receptor that the ligand binds to? 3. What is association? What is dissociation? 4. Write an equilibrium expression for the binding of a ligand to a receptor. Write the equilibrium constant for this reaction. 5. If the equilibrium constant for a ligand binding to a receptor is small, is it correct to say that the receptor will never bind to the ligand? Explain why or why not? 6. What is the role of LCP is determining how much of a free ligand vs. bound ligand is present in a mixture of ligand and receptor. 7. Define affinity and specificity. In what way are these two terms similar and in what way are they different? 8. Dexedrine and Benzedrine are both stimulant class drugs that are prescribed for Attention Deficit Hyperactivity Disorder (ADHD). The chemical and physical properties of the two drugs (elemental analysis, melting point, solubility, NMR, and IR spectra) of the two drugs are identical . The structure of both drugs is shown below; the figure does not , however indicate anything about the actual stereochemistry of either drug. The recommended oral dosage of Dexedrine is 5 mg/day while that of Benzedrine, which has been discontinued, is 10 mg/day. Based on the information provided, give the most plausible explanation as to the differing required dosages of the two drugs.

8. What is the lock-in-key model and what is the induced fit model? Do most biological receptor – ligand interactions resemble lock-in-key or induced fit? 9. If we are comparing the interaction of a receptor with two ligands, L 1 and L 2 , if K A of L 1 is greater the K A of L 2 , which ligand is more likely to be found bound to its receptor? 10. If we are comparing the interaction of a receptor with two ligands, L 1 and L 2 , if K D of L 1 is greater the K D of L 2 , which ligand is more likely to be found bound to its receptor? 11. Why do we express receptor affinities in terms of K D instead of K A ? Write the equilibrium expression for K D . 12. What is meaning of the term fractional saturation? How is fractional saturation defined mathematically? How would we measure the fractional saturation of a receptor binding to a ligand? 13. Show that when [L] = K D , =0.5. 14. Three different proteins can all bind to the organic molecule 2-(4- isobutylphenyl)propanoic acid (ibuprofen). Based on the data in the table below, (a) What is the K D for Protein 2 binding to ibuprofen (include appropriate units)? (b) Which of these proteins binds most “tightly” to ibuprofen (highest affinity)? Ibuprofen (nM)  -Protein 1  -Protein 2  -Protein 3 0.2 0.048 0.29 0.17 0.5 0.11 0.5 0.33 1 0.2 0.67 0.5 4 0.5 0.89 0.8 10 0.71 0.95 0.91 20 0.83 0.97 0.95 50 0.93 0.99 0.98 15. Why is the fractional saturation curve (binding curve) generally hyperbolic ? How can you obtain the K D from a fractional saturation curve? 16. If K D = 20 nM and [L] = 6 nM, calculate the fractional saturation (  ). 17. What type of interactions are responsible for ligands binding to receptors? What is the role of “molecular shape” in determining if a ligand binds to a receptor?

18. What is the role of chirality in determining the specificity of ligand-receptor binding? 19. If K A for the reaction A+B  is small, is most of A found free or as part of a complex? 20. What is the relationship between K A and K D ? Since K A is far more intuitive, why do we measure receptor – ligand interactions using K D ? ( This is a repeat of a question presented earlier; make sure you understand this concept ). 21. Using the fractional saturation curve on the slide titled “Sample Binding Curve”, estimate (do not calculate) the K D . 22. What general class of ligand – receptor interactions is the highest affinity? What general class of ligand-receptor interactions is the lowest affinity? 23. What is allostery? 24. What is cooperativity? Can one have allostery without cooperativity? Can one have cooperativity without allostery? 25. Explain the following terms: heterotropic, homotropic, negative cooperativity, positive cooperativity. 26. What is avidity? How can a high avidity compensate for a low affinity? What are the requirements for a ligand-receptor interaction to have avidity? 27. What are some of the major roles for oxygen in biology? 28. What was the “Great Oxygenation Event” and how did it shape the evolution of life on earth? 29. Why is oxygen required to have mechanically stable organ tissues? 30. Why is a specialized form of gas transport required by multicellular organisms? 31. Describe the gas transport system used by insects. Why is this mechanism potentially insufficient to explain the gas transport system of insects that lived during the Carboniferous? 32. What is the evolutionary origin of lungs? 33. What is unique about the respiratory mechanism of the loach fish? 34. What is Henry’s Law? Why is Henry’s law useful in the study of oxygen binding proteins?

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35. What is heme ? What is the role of heme in myoglobin? 36. Why do we call heme a porphyrin ? Given the structure of a porphyrin, identify the four pyrrole units. 37. Go to the PDB and load the structure 1mbo . Draw the cofactor in the protein. Is the cofactor bound to oxygen in this structure? Go the style option and switch to the space filling representation. Take a screenshot. In this representation, is the oxygen- binding site accessible to the surrounding solvent? What must happen for the protein to for it to bind oxygen? ( Note: the answer to this question is not in the Power Point. You will have to figure out this one on your own ). 38. How do steric effects contribute to the binding of oxygen vs. carbon monoxide by myoglobin? 39. How does the folded polypeptide of myoglobin prevent an unfavorable chemical reaction involving the heme, oxygen, and water? 40. What is the role of the distal histidine in myoglobin in binding to (a) O 2 and (b) CO? 41. Draw the binding curve for O 2 /myoglobin. Why is the curve hyperbolic ? Why can we use the partial pressure of oxygen gas as a surrogate measure of the concentration of dissolved oxygen? 42. What are the major criteria for a good oxygen transport protein? Using the fractional saturation curve of myoglobin, provide a quantitative explanation for why myoglobin would be a poor oxygen transport protein . 43. What is the name our oxygen transport protein? 44. What is the quaternary structure and subunit composition of our oxygen transport protein? 45. Does the subunit of our oxygen transport protein resemble myoglobin? What is the major secondary structure that is present? Is the sequence of the subunits of our oxygen transport protein similar to the oxygen storage protein myoglobin? 46. Why is the fractional saturation curve of hemoglobin sigmoidal? What is the relationship between P50 and the affinity of hemoglobin for oxygen? 47. The subunit composition of hemoglobin is a 2 b 2 . Why is it better to think of hemoglobin as having a subunit composition of (ab) 2 . What experimental evidence supports this alternative way of describing the subunit composition? What is the significance of this experimental evidence as it relates to the function of hemoglobin as an oxygen transport protein.

48. Use the figure below to estimate what percentage of oxygen would be released by hemoglobin that binds oxygen 100 mm Hg pressure and releases the oxygen at 20 mm Hg pressure. 49. Use LCP to explain the binding of oxygen to T-hemoglobin and how this leads to the transition to R-hemoglobin. Then use LCP to explain the dissociation of oxygen from R-hemoglobin and how this leads to the transition from R-hemoglobin to T- hemoglobin. 50. Using the fractional saturation curve of hemoglobin, provide a quantitative explanation for why hemoglobin an excellent oxygen transport protein . 51. What is the meaning of the term “sigmoidal binding curve”? What is the meaning of the term cooperativity ? What is the minimal requirement for cooperativity? 52. What is the structural change in the heme group that occurs upon oxygen binding, and how is this change propagated to the rest of the protomer? 53. What is the major structural difference in hemoglobin between the T and R states? 54. What is the meaning of the term allostery ? What is the meaning of the term homotropic and heterotropic ? Is oxygen a homotropic or heterotropic modulator of hemoglobin? 55. How do allostery and cooperativity contribute to the oxygen transport function of hemoglobin? Consider the role of R-hemoglobin and T-hemoglobin in your answer. 56. Draw the binding curve for O 2 /hemoglobin. Superimpose on this figure the hypothetical binding curve for T-hemoglobin and R-hemoglobin. How do these two hypothetical binding curves account for the sigmoidal binding curve of hemoglobin?

57. What is the Hill equation? What is represented by the term “ n ”? How is this term a measure of the cooperativity in binding multiple ligands to distinct sites on a protein? If a protein such as hemoglobin has 4 binding sites, why is it not possible that n = 4 (absolute cooperativity)? 58. What is the major difference between the concerted and the sequential models of oxygen binding to hemoglobin? As part of your answer, explain both models and how they result in the transport of oxygen from the oxygen rich lungs to the oxygen poor tissues. 59. Explain how the binding of oxygen to heme results in the change in the quaternary structural arrangement in hemoglobin. Include the role of the proximal histidine, helix F, and the  1  1/  2  2 interface. 60. What is the Bohr effect? How does the Bohr effect contribute to the transport of oxygen and carbon dioxide? Illustrate using both text and a binding curve . 61. What is a carbamino acid, and how does this contribute to the transport of carbon dioxide and oxygen in the body ( do not neglect the Bohr effect in this process )? 62. What is the Chloride Shift? How does the Chloride Shift result in the efficient “use” of carbonic acid products as allosteric modulators of oxygen binding to Hemoglobin. What is the experimental evidence for the Hamburger effect? 63. What is the role of 2-BPG in the transport of oxygen by hemoglobin? Illustrate this using both text and a binding curve. Use the concerted model or the MWC model as part of your explanation. 64. How does 2-BPG give rise to high altitude oxygen adaptation? Why does training at high altitude confer some advantage when performing aerobic exercise at sea level? 65. Explain using a fractional saturation curve, why oxygen is preferentially transported from the mother to the fetus . 52. What is the effect of the following changes on the O 2 affinity of hemoglobin? (a) A drop in the pH of blood plasma from 7.4 to 7.2. (b) A decrease in the partial pressure of CO 2 in the lungs from 6 kPa (holdings one’s breath) to 2 kPa (normal). (c) An increase in the BPG level from 5 mM (normal altitudes) to 8 mM (high altitudes). 53. In the lab, it is possible to take hemoglobin and treating it with low concentrations of denaturants, dissociate (break apart) the hemoglobin tetramer (  2  2 ) into a dimer, without disrupting the conformation of the individual protomers. What is the subunit composition of the dimer? Would the new dimer bind oxygen? If so, would the binding

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curve be more like native hemoglobin, R-hemoglobin or T-hemoglobin. Explain your answer. 54. What is the biochemical cause of sickle cell anemia? Despite being associated with a lethal disorder, it continues to persist in a population. Explain why. 55. What adaptation allowed the Wooly Mammoth to leave large portions of its surface exposed to extreme cold? 56. What is the unique physiological feature of the crocodile ice fish ?