Name:______________________________________ Section :______ 7.014 Problem Set 2 Answers to this problem set are to be turned in at the box outside 68-120 by 11:45 am, Friday, February 18. Problem sets will not be accepted late. Solutions will be posted on the web. Question 1 Open the Human γD crystallin (HγD-Crys) structure html file by clicking on the link on the Problem Sets page. Rotate the molecule and get an idea of the three dimensional structure of (HγD-Crys). HγD-Crys is a soluble human eye lens protein. I. The true 3-dimensional structure of HγD-Crys can be observed using the “Show Spacefill” button, and a trace of the peptide backbone can be observed using the “Show Ribbon” button. a) If possible/applicable, describe the following types of structural features of HγD-Crys: i. Secondary: ii. Quaternary (Hint: Use the “Color Ribbon by Protein Subunit” button): b) Does there appear to be a gap between the two protein domains in the ribbon and spacefill structure representations? Ribbon: Spacefill: c) Which of the two representations (spacefill or ribbon) more accurately represents the actual structure of HγD-Crys in the cell? Why? II. We will now look at some interactions important in maintaining the tertiary structure of HγD-Crys. d) Choose “Central Interaction” i. What are the names of the amino acids involved in this interaction? (Hint: It may help to change the representation of the residues to ball and stick by clicking in the box labeled “Ball and Stick on/off” below the 3D window. Keep in mind that in cpk coloring, gray is carbon, blue is nitrogen, and red is oxygen. Hydrogen is not shown!)2 Question 1, continued ii. The central cluster is composed of what type of amino acids (acidic, basic, hydrophobic, or polar)? iii. What type(s) of interaction(s) are formed between residues in this cluster? iv. Select “Central Interaction with All Atoms Shown.” This view shows all the atoms of HγD-Crys (not including hydrogens) as transparent dots. Is the central interaction located on the inside or the outside of the protein? Why is this reasonable given that HγD-Crys is a soluble protein? e) Choose “Interaction I” i. What are the names of the amino acids involved in this inter-domain interaction? ii. What types of amino acids are involved in this interaction (acidic, basic, hydrophobic, or polar)? iii. What type(s) of bonding interaction(s) are found between these two residues? iv. Based on the 3-D structure of amino acid pairs, what parts of the amino acid are involved in forming this interaction (circle the correct answer)? Only the amino acid side chains Only the amino acid backbones Both the amino acid side chains and backbones3 Question 1, continued v. What one amino acid substitution could make this interaction stronger? What one amino acid substitution could make this interaction weaker? Justify your answer in each case. III. HγD crystallin protein is known to be involved in cataract formation in the eye. Cataracts form when several proteins in the eye bond to one another to create a very large insoluble multi-protein complex. There is a point mutation that changes the arginine normally found at position 14 to a cysteine. Children with this mutation develop cataracts. The overall 3-D structure of the protein remains the same despite this substitution. Both in the wild type and this mutant version of the HγD crystallin protein there is also a cysteine at position 110. Click on the button labeled “Residues 14 and 110” to see the location of these two amino acids. f) Are the amino acids at positions 14 and 110 found in the inside or on the outside of the HγD-Crys structure? g) Consider two molecules of mutant HγD crystallin protein. Recall that these molecules would have cysteine residues in both positions 14 and 110. What type of interaction could form between these two molecules? h) How could interaction in part g above lead to the development of a cataract?4 Question 2 The enzyme Phosphofructokinase (PFK) catalyzes the conversion of fructose 6-phosphate (F6P) to fructose 1, 6-bisphosphate (FBP), in step 3 of glycolysis. fructose 6-phosphate + ATP fructose 1,6-bisphosphate + ADPphosphofructokinase ∆Go=−3.4kcalmol a) What two functions does ATP serve in this coupled reaction? b) Draw and label an energy diagram for this reaction. Include the relative energy levels of the substrates and the products, the activation energy and the ∆G° for the reaction. In erythrocytes, the following intracellular concentrations of metabolites are found: Metabolite Concentration Fructose 6-phosphate .014 mM Fructose 1,6-bisphosphate .028 mM AMP 1 mM ADP 0.2 mM ATP 2 mM Pi 1 mM Reaction Progress5 Question 2, continued c) What is the free energy change of the phosphofructokinase reaction under these cellular conditions (37°C)? Show your work. Is the reaction spontaneous under these conditions? Phosphofructokinase (PFK) is the primary site for regulation of the glycolytic pathway. The activity of PFK is dependent on the energy status of the cell. d) Why would a cell regulate glycolysis? e) What is the specific signal or molecule(s) to which PFK responds? f) Why does this mechanism of regulation make sense? g) In certain tumor cells, an enzyme called ATPase becomes abnormally active, resulting in increased hydrolysis of ATP to ADP. What would be the effect on the overall rate of glycolysis in these cells?6 Question 3 The diagram below shows the change in free energy during the course of glycolysis. a) On the diagram, circle the two steps that require ATP. How did you determine that ATP was required for these steps? b) There are steps where the product has a higher free energy than the reactants and yet these steps do not require ATP. What drives these reactions? c) On the diagram, box the energy harvesting steps. d) The harvested energy is stored in what molecule(s)? e) What is/are the molecule(s) listed in (d) used for? G6P— glucose-6-phosphate F6P— fructose-6-phosphate FBP— fructose-1,6-diphosphate G3P— Glyceraldehydes-3-phosphate DAP— Dihydrohy-acetone-phosphate BPG— 1,3-di-phospho-glycerate 3PG— 3-phospho-glycerate 2PG— 2-phospho-glycerate PEP—7 Question 4 Rubisco is a key enzyme in the dark phase of
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