Exam 2 Biochemistry 1 Questions and Answers

EXAM II Biochemistry I I. __________________/ 45 II. __________________/ 26 III. __________________/ 15 IV. __________________/ 14 TOTAL /100 I. MULTIPLE CHOICE. (45 points) Choose the BEST answer to the question by circling the appropriate letter. 1. Which of the following statements about proteins is false? A. B. C. D. E. 2. Which of the following statements about oligomeric proteins is false? A. B. C. D. E. 3. a Ramachandran plot. molecular model building. x-ray diffraction. light microscopy. electron microscopy. A D-amino acid would interrupt an b-strand made of L-amino acids. Another naturally occurring constraint on the formation of an bstrand is the presence of: A. B. C. D. E. 5. All subunits must be identical. Some subunits may have nonprotein cofactors. A subunit may be very similar to other proteins. Some oligomeric proteins can further associate into large fibers. Subunits can exist in dozens or even hundreds of genetic variations. Determination of the precise spacing of atoms within a large protein is possible only through the use of: A. B. C. D. E. 4. Primary structure determines tertiary structure. Globular proteins are generally very compact. Proteins are sometimes conjugated with carbohydrates. Nonpolar amino acid side chains generally are arranged on the surface where they interact with water. Proteins consists of amino acids linked by peptide bonds. a negatively charged Arg residue. a positively charged Lys residue. two Gly residues side by side. a nonpolar residue near the carboxyl terminus. a Pro residue. Which of the following groups correctly shows intrachain hydrogen bonding (denoted by |||) in an a-helix? A. B. C. D. E. -N-H|||H-N-C=O|||H-C-C=O|||H-N-N-H|||H-R-C=O|||O=C- Page 1 interactions between two adjacent hydrophobic Val residues. 7. All of the following are considered "weak" interactions in proteins except: A. B. C. D. E. 8. Which of the following types of bonds or interactions are LEAST likely to be involved in stabilizing the tertiary structure of most proteins? A. B. C. D. E. 9. Prolyl hydroxlyase Tyrosine hydroxylase Carboxypeptidase Y Chymotrypsin Tryptophan oxygenase The role of an enzyme in an enzyme-catalyzed reaction is to: A. B. C. D. E. 11. hydrogen bonds electrostatic bonds hydrophobic interactions disulfide bonds ester bonds Which of the following enzymes plays a direct role in the biosynthesis of collagen? A. B. C. D. E. 10. van der Waals forces. hydrogen bonds. ionic bonds. peptide bonds. hydrophobic interactions. ensure that the product is more stable than the substrate. make the free-energy change for the reaction more favorable. increase the rate at which substrate is converted into product. ensure that all the substrate is converted to product. do none of the above. Which of the following statements is true of enzyme catalysts? A. B. C. D. E. They increase the stability of the reaction product by allowing ionization, resonance, & isomerizations not available to substrates. To be effective they must be present at the same concentration as their substrates. They increase the equilibrium constant for a reaction, thus favoring product formation. They lower the activation energy for the reaction of S to P. They bind to substrates, but are never covalently attached to substrate or product. Page 2 NAME ________________________________ 12. The NEXT four MULTIPLE CHOICE questions refer to the following problem: The protein a1-antitrypsin (a1AT) inhibits the action of the proteolytic enzyme elastase in lung tissue by binding and mimicking the normal substrate. A mutation has been described in which the only change is the substitution of an Arg residue for a Met residue in a1AT. The altered a1AT does not inhibit elastase but has a new property of inhibiting the blood coagulation protein thrombin. The sequences around the active site of a1AT, altered a1AT, and the natural thrombin inhibitor (antithrombin) are given below: a1AT ....Met-Ser-Ile-Pro-Pro-Glu-Leu altered a1AT ....Arg-Ser-Ile-Pro-Pro-Glu-Leu Antithrombin .....Arg-Ser-Leu-Asn-Pro-Asn-Gly 13. Given the information above, which of the following would be the best method to separate a1AT from altered a1AT? (2 pt) A. B. C. D. E. 14. From the information given, all of the following can correctly be concluded EXCEPT: (3 pt) A. B. C. D. E. 15. The number of trypsin cleavage sites is greater in the altered than in a1AT. The number of cyanogen bromide cleavage sites is greater in a1AT than in altered a1AT. Individuals with this mutation might have a bleeding disorder. Antithrombin differs from a1AT only at the active site. Single-residue changes in proteins can alter substrate-binding specificities. Smoker's lungs are often damaged by elastase because the a1AT is inactive. The best explanation for this is that tobacco smoke___. (2pt) A. B. C. D. E. 16. size-exclusion chromatography ion-exchange chromatography isopycnic density gradient centrifugation SDS-polyacrylamide gel electrophoresis affinity chromatography causes the substitution of Met for Arg in a1AT. causes the substitution of Asn for Pro in a1AT. contains oxidants, which chemically modify the Met in a1AT. contains carbon monoxide, which competes with a1AT for binding to elastase. contains nicotine, which serves as a cofactor for elastase. The hexapeptide segment flanking the substitution in altered a1AT is most likely involved in which type of secondary structure: (2 pt) A. B. C. D. E. a-helix parallel b-sheet anti-parallel b-sheet a turn a collagen helix Page 3 NAME ________________________________ II. SHORT ANSWER (26 points) Give a brief answer as directed to each problem or question below. 17. Pauling and Corey showed that in small peptides, four atoms associated with the peptide bond all lie in a plane. Draw a dipeptide of two amino acids, indicate which four atoms are part of the planar structure of the peptide bond, indicate the bond that rotates to change the phi(f) and the psi(y) angles, and indicate the hydrogen bond donor and acceptor atoms if this dipeptide were in an a-helix. (8 pts) 18. Describe the four levels of protein structure and one technique that can be used to determine or measure each type. (4 pts) 19. You have a peptide with the following amino acid composition; 4Asp, 3Gly, 2His, Leu, Phe, 2Val. Draw the likely chromatograph from amino acid analysis using ninhydrin as a colorometric reagent that would have yielded these results. (6 pts) Page 4 NAME ________________________________ 20. A biochemist purifies a new protein using the steps listed below. After each step, he measures total protein and the units of enzyme activity remaining as shown. What are the missing units for Specific activity? For each method, give the basis for the separation. (8 pts) Total Specific Overall protein activity yield (%) Activity BASIS (mg) ( ) (units) 1. Crude extract 100 2,000 0.05 (100) 2. Ammonium sulfate 50 1,000 0.05 50 precipitation 3. Ion-exchange 45 180 0.25 45 chromatography 4. Affinity 12 40 0.30 12 chromatography Are all of these step(s) useful? would you eliminate, and why? If so, which one(s)? Which, if any, III. TRUE/FALSE. (15 points; 1 point each) Choose the BEST answer to the question by circling the appropriate letter. T F 21. The turnover number of an enzyme refers to the number of cycles of binding and catalysis the enzyme goes through per unit of time. T F 22. Enzymes are thought to achieve their high rates of catalysis by lowering the transition state energy barrier for the slow-step in the reaction in going from substrate to product. T F 23. Vitamin C is a necessary for the enzyme responsible for the Schiff base cross-links in collagen. T F 24. The protein a-keratin, found in hair, is an example of a globular protein composed largely of a-helix. T F 25. The Levinthal Problem refers to the incalculable number of different possible conformations that a protein can have when including all possible combinations. T F 26. Carboxypeptidase Y is used for end-group analysis of proteins and peptides. T F 27. The basis by which proteins separate in gel filtration chromatography using Sephadex is by size with the smallest proteins eluting from the column before the larger proteins. T F 28. The formation of a peptide bond is a type of hydrolysis reaction. T F 29. The titration of water helps determine the pKa for the ionizations near the extremes in pH. T F 30. The buffer capacity of histidine is greatest at the equivalence point, after the titration of the carboxylate and before the titration of the imidazole. Page 5 NAME ________________________________ T F 31. The titration of histidine had three ionizable groups, whereas if the amino acid glycine were titrated there would be two ionizable groups. T F 32. The pH meters used to measure [H+] have a special glass tip that is permeable to only protons. T F 33. A multisubstrate, enzyme-catalyzed reaction in which a product is released before all substrates have been bound to the enzyme is an example of a sequential kinetic mechanism. T F 34. A plot of binding data, Y (fraction of total enzyme as ES) vs. [S], that produces a sigmoidal curve indicates that cooperative binding occurs between active sites on the enzyme. T F 35. Use of acid/base catalysis is one strategy employed by enzymes at their active sites to increase the rate of a reaction. IV. PROBLEM. (14 points) Solve the sequence problem with the information given. 36. A small peptide hormone was found to contain amounts of the following amino acids with the given stoichiometries: 3Ala, Arg, 2Cys, Glu, Gly, Lys, Met, Phe, Pro, Trp, Val. Individual samples of the peptide were treated with the following agents with the results noted (where possible Edman degradation was performed with the results of each round given as E#=X): (a) amino-terminal analysis gave two amino acids: Lys and Ala. (b) carboxyl-terminal analysis gave a mixture of Val and Trp. (c) reduction and alkylation followed by peptide separation gave a decapeptide and a tetrapeptide, which was fluorescent (E1=Ala). (d) trypsin: Lys, a tripeptide (E1=Gly, E2=Glu, E3=Val), and a decapeptide (e) cyanogen bromide: a tetrapeptide (E1=Lys, E2=Ala, E3=Phe, E4=homoserine lactone) and a decapeptide (E1=Ala, E2=Cys). (f) chymotrypsin: a tripeptide, and an 11-mer. What is the primary structure of the peptide? (14 points) Page 6 Test Question Multiple Choice 1 2 3 4 5 6 7 8 9 10 11 13 14 15 16 Short Answer 17 Correct Answer D A C E C C D E A C D B D C D Hydrogen bond acceptor O Y + HN 3 C Ca R H N H Ca R F COO H Hydrogen bond donor 18 The structure of the "R" will vary and should correspond to one of the 20 amino acids. In the box the intermediate resonance structure imparts a partial double bond characteristic to the C-N bond, thereby prohibiting rotation. The N and H of the peptide and the C and O of the carbonyl are all in the same plane, or box. The N-Ca is the f-bond and the Ca-Carbonyl is the y-bond. In an a-helix the carbonyl is an acceptor and the NH is a donor. -Primary-Edman degradation, protein sequence analysis, etc. Secondary-Circular dichroism (CD) spectroscopy Tertiary- X-ray crystallography or NMR Quaternary-gel filtration/SDS PAGE, or ultracentrifugation/SDS PAGE Page 1 Test Question Correct Answer -- 19 absorbance or amount Asp Gly Val His Leu Phe fractions -1. Units of Specific activity are units/mg 2. Basis: 1) solubility, 2) charge, 3) binding 3. Yes, step 3, there has been a 5-fold purification relative to prior step and only 5% loss in activity. 4. Both step 2 and 4 are not good. Each has little to no gain in specific activity and anywhere from 50% loss (#2) to nearly 75% loss (#4). Affinity column may not be working because this method is usually quite effective. -- 20 True/False 21 22 23 24 25 26 27 28 29 30 31 32 T T F F T T F F T F T F 33 34 35 F T T (hydroxlyl-proline synthesis) (fibrous) (larger proteins elute before the smaller) (condensation) (also permeable to other salts if high enough concentration) (ping-pong) Page 2 Test Correct Question Answer Problems 36 Lys-Ala-Phe-Met-Ala-Cys|-Arg-Gly-Glu-Val Ala-Cys|-Pro-Trp After experiments (a-c) you should realize that there are two peptides joined by a disufide with the 2 Cys residues. The fluorescence puts the Trp on the C-term of the smaller 4-mer with the Val then on the C-term of the 10-mer. The GEV sequence of the trypsin puts this at the other C-term with the R adjacent and the K at the N-term of the 10-mer with the Ala then at the N-term of the 4-mer. CNBr gives more of the 10-mer; KAFM, but the AC of the decapeptide means that BOTH N-term must be Ala-Cys and that only leaves Pro as third residue in the 4-mer. -- Page 3