7.014 Problem Set 2 Solutions

Transcription

1 7.014 Problem Set 2 Solutions Please print out this problem set and record your answers on the printed copy. Answers to this problem set are to be turned in at the box outside by 11:45 Friday, February 24. Problem sets will not be accepted late. Solutions will be posted online. Question 1 You recently started a UROP working in the lab of Dr. Ami No. One of the members of her lab has recently discovered, purified, and crystallized a new protein, Prot1, whose structure is shown below. a) What kind of secondary structure is illustrated in green? Beta sheets b) What kind of secondary structure is illustrated in red? Alpha helices Dr. No is particularly interested in understanding how this protein folds. In particular, she suspects that the interaction between Asp107 and Arg23 is important in determining the structure of Prot1. c) What type of amino acid is Asp (acidic, basic, hydrophobic, or polar)? Acidic What type of amino acid is Arg (acidic, basic, hydrophobic, or polar)? Basic d) What is the strongest interaction that can form between Asp107 and Arg23? Choose from: covalent, ionic, hydrogen bond, and van der Waals. Ionic bond 1

2 Question 1, continued In order to study the folding of Prot1, Dr. No asks you to create several different Prot1 mutants and examine their structures. One of these mutants has a Lys substituted in at the Arg23 position. e) What type of amino acid is Lys (acidic, basic, hydrophobic, or polar)? Basic f) Would you expect this substitution mutation (Arg23 Lys) to seriously disrupt the folding of the Prot1 protein? Why or why not? We wouldn t expect this substitution to significantly disrupt the folding of Prot1because while the shape of Arg and Lys is somewhat different (with Arg having more bulk), Lys can still form the ionic bond with Asp. Consequently, we would expect the 3D shape of the protein to largely remain the same. Next you study a Prot1 mutant where Arg23 has been replaced by Glu. You discover that this mutant is unable to fold properly, rendering the protein unable to function. g) What type of amino acid is Glu (acidic, basic, hydrophobic, or polar)? Acidic h) How does this amino acid substitution cause the protein to fold incorrectly? The mutant protein has an acidic residue where the wild type protein has a basic residue. Therefore, the mutant protein can t form the ionic bond that the wild type protein forms. In fact, the two acidic residues repel each other, resulting in the changed fold of the mutant protein. You find another mutant that also as the same Arg23 Glu mutation. However, this mutant protein is able to fold normally. Intrigued by this discovery, you decide to examine this mutant more closely and discover that it has acquired a second substitution mutation at the Asp107 position. i) What amino acid(s) could be substituted for Asp107 that would allow Prot1 to retain its proper shape and function? Justify your answer. We would expect that the second mutant was able to reconstitute the ionic bond between amino acids in positions 23 and 107. Since the amino acid in position 23 is now acidic, we would expect the new amino acid in position 107 to be basic. There are three basic amino acids His, Lys, and Arg, and, depending on how densely the protein is packed, one or any of these substituted in position 107 should allow the mutant protein to regain the shape close to that of the wild type protein. Question 2 Open the Lysozyme structure html file in Internet Explorer. Rotate the molecule and get an idea of the three dimensional structure of Lysozyme. Lysozyme is a soluble human protein used in the breakdown of cell walls. 2

3 Question 2, continued a) Human cells do not have cell walls. What purpose might lysozyme serve in the human body? Lysozyme is used to make holes in the cell walls of plant and fungi cells during digestion. We need to make holes in the cell wall so we can extract and digest the usable molecules inside. Cells of what organ(s) are likely to produce the enzyme? We would expect the cells lining the stomach to make lysozyme and to secrete it into the stomach. I. The 3-dimensional structure of lysozyme can be observed using the Show Spacefill button, and a trace of the peptide backbone can be observed using the Show Ribbon button. In the CPK coloring scheme that is used here, gray is carbon, blue is nitrogen, and red is oxygen. Hydrogen is not shown. b) Describe the quaternary structure of lysozyme (Hint: Use the Color Ribbon by Protein Subunit button). There is only one amino acid chain, so no quaternary interaction. c) Does there appear to be a gap between the α-helices in the ribbon structure representation? Yes d) Does there appear to be a gap between the α-helices in the spacefill representation? No e) Which of the two representations (spacefill or ribbon) more accurately represents the overall shape of lysozyme in the cell? Why? The spacefill model is a more accurate representation of what lysozyme looks like in the cell. The spacefill representation shows the Van Der Waal radius or electron density clouds of all nonhydrogen atoms in the protein. The ribbon structure simply shows a trace of the polypeptide backbone without showing any amino acid side chain atoms. II. Next bunch of questions looks at some interactions important in maintaining the tertiary structure of Lysozyme. Choose Interaction I f) What amino acids are 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. Also, pausing a cursor over a representation of any atom for 3 seconds will cause the label with the name of the amino acid to which the atom belongs to pop up. The label will also contain the number of the amino acid in the primary structure of the protein.) There are two tryptophans, a leucine, an isoluceine, an alanine, and a valine. g) What type(s) of amino acids are involved in this interaction (acidic, basic, hydrophobic, or polar)? All of these amino acids are hydrophobic. 3

4 Question 2, continued h) What is the strongest type of interaction found between these residues? Hydrophobic effect Select Interaction I with All Atoms Shown. This view shows all the atoms of lysozyme except for those involved in Interacion I (not including hydrogens) as transparent dots. i) Describe the location of this interaction in relation to the outside of the protein. Why is this reasonable given that Lysozyme is a soluble protein? These amino acids are primarily found on the interior of Lysozyme. There are a few areas that are solvent exposed, but most of the cluster is shielded from solvent. It is energetically favorable for this cluster to be hidden from a hydrophilic solvent like the cytoplasm or water because it the amino acid side chains are hydrophobic. Choose Interaction II j) What amino acids are involved in this interaction? An aspartic acid and a glutamine are involved in this interaction. k) What type of amino acids are the residues in this cluster (acidic, basic, hydrophobic, or polar)? Glutamine is polar. Aspartic acid is acidic. l) What is the strongest type of bonding interaction found between these two residues? These two amino acids form a hydrogen bond. III. There are diseases caused by mutations in wild-type lysozyme. These mutations cause the protein to alter its structure and affect the solubility and functionality of the molecule. These types of mutations in lysozyme cause amyloidogenesis (protein aggregation) diseases. In one such mutation, the native residue at position 56, Isoleucine, is mutated to threonine. The mutant protein does not fold up correctly when produced and multiple mutant lysozyme molecules begin to sick together. Turn on Isoleucine 56 by clicking on Residue 56. m) Is residue 56 located on the surface or in the interior of the protein? Residue 56 is located in the interior of the protein. n) What type of residue is isoleucine (acidic, basic, hydrophobic, or polar)? Isoleucine is nonpolar. What about threonine? Threonine is polar. o) How might this change lead to the inability of lysozyme to fold to its native 3-dimensional shape? If Isoleucine 56 is found within a hydrophobic pocket on the interior of the lysozyme, replacement of it by a polar residue may disrupt the hydrophobic cluster. The other nonpolar residues may not be able to collapse together to form the hydrophobic core the way they did before, because of the unfavorable OH group now in the pocket. Without proper folding of the core, the molecule may not be able to form around it. 4

5 Question 2, continued In another such mutation, the native residue at position 67, Aspartic acid, is mutated to histidine. The mutant proteins are altered in their primary and quaternary structures, but retain their overall tertiary structure. Turn on Isoleucine 67 by clicking on Residue 67. p) Is residue 67 located on the surface or in the interior of the protein? Residue 67 is located on the surface of the protein. q) What type of residue is aspartic acid (acidic, basic, hydrophobic, or polar)? Aspartic acid is acidic. What about histidine? Histidine is basic. r) How could this mutation effect primary and quaternary structure without affecting the protein s tertiary structure? Lysozyme is a soluble protein. Therefore, a mutation of an acidic residue on the surface of the protein to a basic residue may not change the fact that water interacts favorably with the molecule, and the mutant can stay soluble and may keep the overall tertiary structure. However, a positive charge on the outside of the protein where there was once a negative charge could change the way the protein interacts with other lysozyme molecules or other binding partners. For example, there may be another protein in the body that can now stick to the new positive charge on lysozyme. Question 3 Ignatius Maloy Thursdy, a longtime employee of a large bourbon manufacturer, decides to open up a bourbon microbrewery of his own. To perfect the recipe, he sets up several fermentations in his basement, identical in every way to the open-vat fermentations carried out in the brewery where he works, except his vats are 250 ml, rather than the several thousand liters as at the brewery. a) Iggy worries that something is wrong when he observes yeast growing in the samples much faster than at the brewery. When the yeast stops growing, he finds that no ethanol has been produced, hence no bourbon. Why did Iggy s fermentations fail? In the small vessels, O 2 was readily available for the growing yeast, and rather than ferment glucose to ethanol, they used the pyruvate for respiration. b) Dismayed by his failure, Iggy decides to go back to school to study the biochemistry of the enzymes involved in glycolysis (see p.9 for the pathway). He s fascinated by the enzyme triose phosphate isomerase, because the reaction it catalyzes (DHAP G3P) proceeds so quickly in the cell. He reasons that since this reaction occurs so quickly in the cell, it would probably occur very quickly in the absence of enzyme as well. i) Is his reasoning sound? Explain. No, Even if the reaction is thermodynamically favorable, it may not occur due to the activation energy ( G or E a ). Without enzyme, the high activation energy can prevent the reaction from proceeding on appreciable scale. 5

6 Question 3, continued b) ii) This reaction is truly reversible and the reverse reaction is equally as likely as the forward reaction. Explain why the reverse reaction, G3P DHAP seldom occurs in the cell. G3P is rapidly removed in the next step of glycolysis. This drives the reactions in the forward (DHAP G3P) direction. c) Iggy needs your help. For both i) and ii) fill in the blank with the appropriate response and explain. i) The Gibbs free energy of Glucose would be Glucose-6-phosphate. higher than lower than the same as Explain. Adding phosphate groups to glucose to form Glucose-6-phosphate requires ATP. ii) The Gibbs free energy of Phosphoenolpyruvate would be Pyruvate. higher than lower than the same as Explain. We know from glycolysis that this reaction produces ATP, i.e. that some energy released in this reaction can be stored in the energy storage molecule. iii) Below, draw the energy diagram of the reaction that converts phosphoenolpyruvate to pyruvate. Include all the reactants and products. On the diagram, label G and E a ( G ). E a ( G ) PEP +ADP G pyruvate +ATP 6

7 Question 5 Alcohol dehydrogenase is the enzyme that process alcohol in our bodies. An individual s alcohol tolerance is largely dependent upon the efficiency and concentration of this enzyme within the bloodstream. You isolate the alcohol dehydrogenase from the blood samples of two separate individuals (A and B) whose alcohol tolerances are particularly low. You determine that no matter how much substrate you provide to a given quantity of the alcohol dehydrogenase of individual A, the reaction does not reach the same speed as the reaction catalyzed by the wild-type alcohol dehydrogenase. However, individual A s enzyme has exactly the same affinity for the substrate as the wild-type enzyme. a) What specific defect causes Individual A to have alcohol tolerance lower than that of most people? Justify your answer. The likely defect in individual A s alcohol dehydrogenase is somewhere in the active site. The enzyme has the same affinity for the substrate as the wild type but can t operate as fast as the wild type enzyme. Therefore, the defects is in the inability to perform as many reactions per unit time as the wild type enzyme. Thus, we conclude that some part of the enzyme active site mechanism is not operating as efficiently as that of the wild type enzyme. On the other hand, the alcohol dehydrogenase of Individual B behaves exactly the same as the wild-type alcohol dehydrogenase. b) What is a likely reason for why Individual B has a lower alcohol tolerance than most people? Justify your answer. The enzyme from Individual B performs the same as the wild type enzyme. Therefore, we must conclude that this individual s low alcohol tolerance must be due to the low amount of enzyme produced by his cells. 7