Name: Chem 351 Exam 1

Transcription

1 Use the pk a values below for all problems requiring pk a s: α-amino groups = 9.4 α-carboxyl groups = 2.3 Sidechain ionizable groups: Cysteine = 8.5 Tyrosine = 10 Lysine = 10.5 Arginine = 12 Histidine = 6.8 Glutamate/Aspartate = 4.0 Serine/Threonine = 13.5 Multiple Choice For Problems 1-15, pick the BEST answer. Write the letter corresponding to your choice in the box provided at the end of this section (2 points each). 1) In the very simple Michaelis-Menten reaction scheme, k 2 is: A) assumed to be 0 in order to make the mathematical model easier to interpret. B) part of the rate equation that expresses the rate of formation of ES. C) the rate constant for the rate determining step of the chemical transformation. D) the highest rate constant in the reaction scheme. E) None of the above statements is correct. F) More than one of the above statements is correct. 2) Which of the following statements would be TRUE when comparing the titration curve for the amino acid V to the titration curve for the amino acid Y? (The titration curves are followed from ph 0 to ph 14; V and Y are present at the same concentration). A) The titration curve for Y would contain longer buffer zone(s) than the one for V. B) The titration curves for V and Y would have very similar pi points. C) The titration curve for V contains only one buffer zone. D) The titration curve for Y contains more inflection points than the titration curve for V. E) None of the above statements is true. F) More than one of the above statements is true. 3) Which of the following accurately describes the molecular basis for muscle activity? A) ATP hydrolysis forces the movement of the thin filament relative to the thick filament. B) ADP and P i are released together, in the same molecular event. C) ATP binding causes the thick and thin filaments to dissociate. D) Calcium is released from troponin when a nerve stimulates muscle contraction. E) When ATP is not available at all, the actin-myosin association cannot form. 4) Which of the following statements about structural proteins is false? A) Keratin is composed of two polypeptides with their α-helices twisted around each other. B) Collagen contains three polypeptides with their α-helices twisted around each other. C) Fibroin is predominantly composed of β-sheet in antiparallel orientation. D) Covalent bonds often connect different polypeptides in collagen and keratin. E) Gly is a common amino acid found in collagen and fibroin. 5) Enolase: A) is an enzyme that moves a phosphate group from ATP to an inorganic substrate molecule. B) uses magnesium ions to accept electrons in a redox reaction. C) uses a Lys sidechain located in its active site as a base. D) catalyzes the hydrolysis of a peptide bond. E) None of the above. 1

2 6) As it applies to hemoglobin, the Bohr effect: A) shows how pressure changes impact O 2 binding. B) is best demonstrated by a rightward shift of the oxygen binding curve as ph decreases. C) explains the increased carbamoylation of hemoglobin where and when the ph is high. D) is used to explain how hemoglobin can pick up more O 2 at high altitude. E) does all of above. 7) Proteins A and B have vastly different physical properties in their native states. Protein A has a pi of 3 and a M r of 97 kda, while B has a pi of 8 and a M r of 39 kda. What can you conclude about Proteins A and B? A) In a cation exchange column run at ph 7, Protein B should elute first. B) In reducing SDS-PAGE, Protein A s band should migrate farther. C) In size exclusion (gel filtration) chromatography, Protein B should elute first. D) In an anion exchange column run at ph 7, Protein A should bind more tightly. E) None of the conclusions above can be made. F) More than one of the conclusions above can be made. 8) A membrane bound sac containing 35 mm Na 2 HPO 4 is dropped into a beaker that contains a solution of 20 mm KCl and 20 mm CaSO 4. Which of the following statements is true? A) The two solutions are isotonic. B) The solution in the sac is 70 milliosmolar. C) The sac will soon shrivel. D) The solution in the beaker is 40 milliosmolar. E) None of the above is true. 9) A short polypeptide has sequence H-W-F-M-A-V-K-R-Y-P. In the process of sequencing the polypeptide, it was treated with a variety of chemical agents and digested by several proteases. Which of the following results would have been predicted or expected, assuming successful and complete treatments? A) Cyanogen bromide treatment yielded three fragments of varying lengths. B) Trypsin treatment yielded three fragments of varying lengths. C) Chymotrypsin treatment yielded three fragments of varying lengths. D) FDNB treatment yielded only a 2,4-dinitrophenyl derivative of His. E) None of the above would have been expected. F) All of the above would have been expected. 10) What does [ES] represent in Michaelis-Menten kinetics? A) It represents the number of substrate molecules added to the assay. B) It is a constant that is independent of substrate concentration. C) It is equal to [E total ] when v 0 = V max. D) It is equal to ½ V max. E) None of the above is true. 11) Antibodies/immunoglobulins: A) can be detected based on their native enzymatic activity. B) would appear as multiple bands in native PAGE. C) actively digest invading proteins via their inherent protease activity. D) are susceptible to disulfide reduction. E) are able to bind numerous antigens in both their Fab and Fc domains. 2

3 12) Which of the following best describes the structure/function of myoglobin? A) Myoglobin is an oxygen binding protein that demonstrates a classic rectangular hyperbola binding curve. B) Each myoglobin protein binds to several hemes, allowing enhanced O 2 binding ability and a better ability to store O 2. C) Myoglobin s primary sequence is similar to hemoglobin s sequence, but myoglobin s quaternary structure is essential for its high binding affinity. D) According to oxygen binding curves, myoglobin is able to drop off O 2 effectively, even when po 2 is high. E) Myoglobin demonstrates cooperative binding in order to pick up and store O 2 in tissue more effectively than hemoglobin does. 13) Phosphoric acid (H 3 PO 4 ) is a polyprotic acid with pk a s of 2.14, 6.86, and What is the net average charge on all of these molecules (consider the form or forms that predominate) when a solution of phosphoric acid is sitting at a ph of 4? A) +2 B) -1.5 C) -1 D) +1.5 E) None of the above. 14) The presence of increasing amounts of 2,3-bisphosphoglycerate (BPG): A) leads to an O 2 binding curve for oxygen that is increasingly shifted to the left. B) significantly enhances the ability of O 2 binding when hemoglobin is moving through the lungs. C) significantly decreases the affinity of hemoglobin for O 2. D) affects fetal hemoglobin more strongly than the mother s hemoglobin. E) more than one of the above is true. F) none of the above is true. 15) Which of the following statements about secondary structural motifs is false? A) The α-helix is a regular arrangement of residues in which sidechains project outward, away from the center of the helix. B) In antiparallel β-sheets hydrogen bonding occurs between N-H and C=O groups of the polypeptide backbone. C) β-turns often involve a stretch of four residues, with a proline residue in the second position. D) The pitch of an α-helix is 3.6 Å/turn. E) None of the above is false

4 Short Answer. Show your work for all calculations! 1) Provide the missing information. (1 or 2 points each): 1-letter 3-letter 3-D structure at ph 7 pi (circle one) K < 6 > 6 ~ 6 Asn < 6 > 6 ~ 6 T < 6 > 6 ~ 6 2) Consider an arginine residue buried in a protein s interior. How would the pka of arginine s sidechain be expected to shift (circle the most likely choice) if the group closest to it in space was changed (2 pts each): a) from a lysine sidechain to a glutamate sidechain? pka rises pka falls pka is unchanged b) from an I sidechain to a L sidechain? pka rises pka falls pka is unchanged c) from a Val sidechain to a His sidechain? pka rises pka falls pka is unchanged 3) Now consider a salt bridge within the hemoglobin protein. a) What equation defines the magnitude of force holding this bridge together? Define all terms. (4 points) b) How could this force holding the salt bridge together change if the salt bridge moved into an environment that contained only hydrophobic residues and decreased water accessibility? Explain by addressing ALL the things in your equation that could be impacted (which terms?) and state how they would change (increase, decrease). (2 points) 4

5 4) Name: Chem 351 Exam 1 In the figure above, label a) A complete sarcomere b) G actin c) Myosin head d) I band e) A band f) M line 5) Several different antibodies were isolated from a polyclonal production. One binding study examined Antibody Y s affinity for antigen D, and the graphical results are shown at right. a) Estimate K d and K a for this antibody/antigen relationship. Show exactly how you are choosing your values. (3 pts) θ Ab y [antigen D] mm b) Calculate (do not simply estimate it from the graph) θ when [antigen D] = 1.72 mm. Write the equation that you must use to make this calculation. (3 pts) c) A different antibody (Ab z ) demonstrates a slightly larger K d for the same antigen. Draw a reasonable binding curve for Abz in the plot above. (2 pts) 5

6 6) The observed acetylation of a serine residue in the active site of COX-2 by aspirin can be predicted to start with a simple nucleophilic attack by serine s sidechain, aided by a proton transfer. Given the hypothetical environment (all sidechains shown participate directly in the mechanism), provide a curved-arrow mechanism for this step and draw all products (charges should balance) that form (hint: an oxyanion hole is observed to form during this step, just as it does in chymotrypsin s acylation step). (12 points total) aspirin O acetyl group O OH H O Ser N H N His 7) Assume that an enzyme activity was monitored in the presence or absence of each type of inhibitor given below (all other conditions and quantities remaining the same). For each type of inhibitor, write the letter(s) of the observations (A-I) that would apply. Some descriptions may be used more than once, and some may not be used at all. (3 points each box) INHIBITOR: UNCOMPETITIVE MIXED A) Inhibitor binds E. B) Inhibitor binds ES. C) Inhibitor binds at active site. D) Inhibition is irreversible. E) V max decreases with inhibitor F) k cat decreases with inhibitor G) K M decreases with inhibitor H) Slope of LB (double-reciprocal plot) increases typically I) Y-intercept of LB (double-reciprocal plot) decreases typically 6

7 8) Henderson-Hasselbalch. The popular buffer Tris is shown on the right in its fully protonated form. Its pka is Answer the following questions about this buffer. (10 pts total) HO A) What is the ratio of protonated to deprotonated forms of Tris buffer that exist when the solution has a ph of 7.21? HO NH 3 pka 8.07 OH B) What percent of the buffering species is in a charged (+1) form at a ph of 8.35? C) Under what environmental conditions (i.e. at what ph) will the average charge on the buffering species be +0.75? D) If the 100 ml of 0.2 M Tris solution starting at a ph of 7.8 is brought to a ph of 7.4 using HCl, how many moles of buffer will actually change charge during this ph change? 9) Protein Analysis: Digestion of a short polypeptide with trypsin yielded three fragments as shown below. Treatment of the original polypeptide with cyanogen bromide yielded a tripeptide and a hexapeptide. Chymotrypsin digestion of the original polypeptide yielded a tetrapeptide and a pentapeptide. (3 pts each) Trypsin Fragments: T1: Met-Val-Phe-Lys T2: His-Lys T3: Asp-Gly-Lys A) From all of this information above, provide the sequence of the original nonapeptide: B) If these three trypsin fragments were loaded onto a cation exchange column run at ph 6 and eluted with an increasing gradient of NaCl, in what order would they elute? C) What is the pi of fragment T2 (remember that relevant pka values are on the first page)? 7

8 Protein Purification The following short answer questions are about protein isolation methods. 1) Two-dimensional gel electrophoresis was performed on a mixture of proteins. Isoelectric focusing was used first, then a native-page further separated the proteins in the second step (electrodes are labeled for each dimension). The results are shown at right. Based on these results: (2 pts each) a) which of these proteins has the highest pi? A B C D E Cannot tell b) which of these proteins has the highest native molecular weight? A B C D E Cannot tell c) which of these proteins is most likely to be multimeric? A B C D E Cannot tell d) which of these proteins would be the last to elute from a gel filtration (size-exclusion) column? A B C D E Cannot tell M r A B 1-D E C D 2-D e) The protein band identified as B in the 2-D analysis above was subjected to SDS-PAGE. Two distinct bands (B1 and B2) were identified with the apparent molecular weights listed. Interpret this result with as much detail as possible (4 pts). Band M r (kda) B1 ~75 B2 ~35 2) Anion exchange chromatography was performed as part of a long purification procedure. During this step proteins were bound to the column, washed with buffer at ph 7, then eluted using a gradient of increasing KCl concentration. The protein of interest eluted last, in Pool 3. Use the information in the table to: a) Calculate the specific activity of this protein in Pool 3 (3 pts). Total Protein Total Activity (mg) (U or µmol/min) Loaded onto the column Pool b) Calculate the enrichment in protein purification that occurred in this step (3 pts). c) Compared to the pools of protein that eluted earlier (Pools 1 and 2), Pool 3 s pi is (2 pts): Higher Lower Impossible to determine 8

9 Kinetics and Activity Kinetic analysis was performed on an enzyme known as Blue. Catalytic activity was measured as a function of substrate [maize] concentration. υ 0 [S] was given in mm concentrations v 0 s were measured in µmol/sec 5 µg of enzyme was used in each assay Each assay was 1mL in total volume M r Blue is 56 kda Blue [maize] mm Define each term and estimate their values using the graph above: (4 pts ea) A) K M : B) V max : C) k cat : D) What would happen to the Blue activity in the presence of crimson, a competitive inhibitor? 1) Explain, and draw the expected activity curve in the plot above (2 points). 2) A Lineweaver-Burk (double-reciprocal) plot was made for the kinetic data above. Using your estimates above, determine the following numbers for the L-B plot, and indicate those values that would change in the presence of crimson. (6 pts) In the LB plot Value Crimson would change this value Y-intercept X-intercept Slope 9

10 Catch-all Fun 1) Consider the perspective drawing for a short section of polypeptide shown below. a) Identify all complete amino acids in this polypeptide by circling each and providing 3-letter abbreviation(s). b) Which bond rotation leads to changes in the ψ (psi) dihedral angle? - label the(se) bond(s) on the figure. c) Now choose the bond whose rotation represents the φ (phi) dihedral angle for the first residue shown in this fragment. Draw a Newman projection for this bond, using the α-c as the front atom. You may abbreviate your chain, but clearly show all atoms or groups (including the sidechain) directly attached to the bond being rotated. Chain N H CH 2 SH C H N O C Chain O S Hemoglobin and Myoglobin (1pt each): NOT YET FINISHED? a) Carbomoylation of hemoglobin is more common in the lungs where carbon dioxide is freely available. b) Myoglobin has quaternary structure. c) At the lungs, the concentration of BPG is a significant factor because it lowers the amount of oxygen can be picked up by 50%. d) O 2 binding is the most important factor in switching T hemoglobin into R hemoglobin. e) Myoglobin s binding affinity (for O 2 ) is significantly higher than hemoglobin s binding affinity at nearly all po 2 observed in tissue. Multiple Choice /30 Page 6 /18 Page 9 /20 Page 4 /24 Page 7 /19 Page 10 /10 Page 5 /14 Page 8 /20 Total /150 (155) 10