Final exam 2016, questions PDF

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BIOC*2580 Final Examination Winter 2016 Thursday, April 21st, 2016, 2:30 – 4:30 p.m. PLEASE PRINT YOUR NAME HERE LAST NAME: ___________________________________ FIRST NAME: ________________________________ STUDENT NUMBER: __________________________ REMEMBER TO ENTER YOUR NAME AND STUDENT NUMBER ON THE “SCANTRON” CARD! (Your e-mail address is not required.)

Instructor: Dr. Enoka Wijekoon Time allotted = 2 hours (120 minutes). This is a closed-book exam: no notes or aids of any kind (other than a calculator with no stored information) may be consulted. This booklet has 17 pages, including the metabolic chart. Total marks for this paper = 80 This examination determines 40% of the final course grade. Multiple-choice questions. 40 questions; 40 marks total; no marks will be deducted for incorrect answers. Use a soft pencil to mark your answers on the scantron sheet. In the event of a discrepancy between the scantron sheet and what has been noted on the exam booklet, the RESULTS ON THE SCANTRON ARE TAKEN AS FINAL. Written answer questions. Answer all questions. 40 marks total.

Notice: No marks will be given for illegible and otherwise unreadable, incomprehensible, or unclear answers or writing. (Do not write below this line)

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Multiple-choice questions. Forty (40) questions = 40 marks total. For each question, choose the best answer from among the possible answers given. Enter your answers on the Scantron card. 1. Which of the following amino acids is a disruptor or breaker of secondary structure? a) Ile b) Asp c) Ala d) Glu 2. Which of the following amino acids will bind to anion exchange resin at pH 7.0? a) a) Asp b) Asn c) Lys d) Leu 3. Given that it takes approximately 14 turns of an alpha-helix in a membrane protein to span a cell membrane, what would be the average thickness of a cell membrane? a) 21 Å b) 48 Å c) 50 Å d) 76 Å 4. The attacking nucleophile that starts the catalytic process in chymotrypsin is: a) Aspartate b) Histidine c) Serine d) Tyrosine 5. What initial reaction velocity (v0) is observed if substrate concentration in an enzyme reaction is 0.5 x KM and Vmax is 2.4 x 10-6 mol L-1 min-1? a) 1.2 x 10-6 mol L-1 min-1 b) 6.0 x 10-7 mol L-1 min-1 c) 2.4 x 10-6 mol L-1 min-1 d) 8.0 x 10-7 mol L-1 min-1 6. Two-dimensional gels make use of two separation methods. These two methods are... a) Thin layer chromatography and ion exchange chromatography b) Thin layer chromatography and SDS electrophoresis Page 2 of 23

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c) Isoelectric focusing and SDS electrophoresis d) Isoelectric focusing and ion exchange chromatography 7. Which of the following is NOT true about SDS-polyacrylamide gel electrophoresis of proteins? a) It separates proteins that differ in molecular weight b) SDS denatures the proteins c) An electric field is used to move the proteins through the gel d) Larger proteins move through the gel faster than smaller proteins 8. Which of the graphs shown on the right best represents the initial velocity (v0) of the reaction as a function of substrate concentration [S]? a) A b) B c) C d) D 9. Which of the following best describes the type of reaction catalyzed by a kinase? a) oxidation b) hydrolysis c) phosphorylation d) decarboxylation 10. When two carbohydrates are epimers: a) one is a pyranose, the other a furanose b) one is an aldose, the other a ketose c) they differ in length by one carbon d) they differ only in the configuration around one carbon atom 11. Which of the following alcohols is not commonly found as a constituent of glycerophospholipids? a) Choline b) Ethanolamine c) Serine d) Threonine 12. At neutral pH, the net charge on the DNA polynucleotide strand 5′ TAACGCT 3′, bearing OH groups at both the 5′ and 3′ ends, will be: a) -10 b) -6 c) 0 Page 3 of 23

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d) +6 13. The pattern of double bonds in commonly occurring polyunsaturated fatty acids is best represented as: a) –CH2-CH=CH-CH2-CH=CHb) –CH2-CH=CH-CH2-CH2-CH=CHc) -CH2-CH=C=CH-CH2 d) –CH=CH-CH=CH14. Which of the following statements about nucleic acid structure is false? a) Ribose is the RNA sugar that corresponds to the DNA sugar deoxyribose. b) The double helix is formed from two antiparallel right handed helical strands of DNA. c) A phosphate group bridges between the 5′ OH of one nucleotide unit and the 3′ OH of another forming a phosphoanhydride linkage. d) The % GC content of bacterial DNA samples varies substantially among species. 15. The structure shown at right represents a “generic” sugar molecule; the chiral configurations are not specified. Corresponding to this structure, there are X possible stereoisomers, Y of which are “D” sugars. The values of X and Y are…. a) 8 and 4 b) 16 and 8 c) 12 and 6 d) 6 and 6

HC = O C(H)OH C(H)OH C(H)OH CH2OH

16. Which of the following factors contribute to the large negative energy release (-ΔG) associated with the hydrolysis of ATP? a) Electrostatic repulsion among the negative charges of ATP b) The greater resonance stabilization of ATP compared to the product Pi c) Ionization of the Pi product of the reaction to HPO42- from H2PO4d) All of the above 17. Give the correct answer about ATP utilization. a) A 70 kg person uses about 4 g ATP during a “restful” day b) A 70 kg person uses about 40 kg ATP during a “restful” day c) The typical lifetime of an ATP molecule in cells is measured in minutes to hours d) The typical lifetime of an ATP molecule in cells is measured in hours to days 18. Transport of fatty acids across the inner membrane of the mitochondrion depends on conjugation to: a) ubiquitin Page 4 of 23

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b) coenzyme A c) creatine phosphate d) carnitine 19. The

cofactor shown below:

a) Acts as an oxidizing agent in redox reactions b) Acts as an electron carrier in the electron transport chain c) Acts as a carrier of acyl groups d) Acts as a reducing agent in biosynthetic reactions 20. Complex III of the mitochondrial electron transport chain… a) catalyzes the reduction of cytochrome c by NADH b) catalyzes the reduction of coenzyme Q (in the quinone form) by NADH c) catalyzes the reduction of cytochrome c by ubiquinol (QH2) d) catalyzes the translocation of two protons across the inner mitochondrial membrane per NADH oxidized 21. All of the following metabolic processes occur primarily in the mitochondria of eukaryotic cells, except; a) Oxidative phosphorylation b) The Krebs (citric acid) cycle c) Beta oxidation of fatty acids d) Glycolysis 22. Which one of the following experimental observations can be explained by the cyclical nature of the Krebs cycle? a) Pigeon breast muscle has a very high rate of oxygen consumption b) Addition of small organic acids like succinate and malate to a pigeon breast muscle preparation resulted in a catalytic increase in oxygen consumption c) Knoop discovered that citrate could be converted to succinate d) Malonate inhibits succinate dehydrogenase leading to the accumulation of succinate 23. According to the mechanism of ATP synthase catalysis proposed by Paul Boyer, the energy released by the proton motive force is used mainly to promote the….. Page 5 of 23

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a) Binding of the α subunit to the β subunit b) Binding of ADP to the enzyme c) Condensation of ADP with inorganic phosphate to form ATP d) Release of ATP from the enzyme 24. The Cori cycle describes: a) The generation of lactic acid in the muscles, and its conversion back to glucose in the liver b) The production of α-ketoglutarate from amino acids, and its conversion back to amino acids c) The mechanism for the passage of protons through the mitochondrial ATP synthase d) The breakdown of proteins in the muscles, and their regeneration in the liver 25. The following four enzymes catalyze the steps of fatty acid β-oxidation: 1. β-hydroxyacyl CoA dehydrogenase 2. Thiolase 3. enoyl CoA hydratase 4. acyl CoA dehydrogenase. What is the order in which they act? a) 4, 2, 1, 3 b) 3, 1, 4, 2 c) 4, 3, 1, 2 d) 1, 4, 3, 2 26. Identify the incorrect statement regarding the malate aspartate shuttle: a) It is involved in transferring NADH- reducing equivalents from the cytosol to the mitochondrial matrix b) It generates a mitochondrial FADH2 for each cytosolic NADH c) The TCA cycle enzyme malate dehydrogenase plays a role in this shuttle d) It makes use of transporter proteins to transport malate into the mitochondria and aspartate out of it 27. The most effective analytical technique for the separation of fatty acids is… a) Gel filtration chromatography b) Ion exchange chromatography c) Gas liquid chromatography d) Thin layer chromatography 28. Conversion of NAD+ to NADH is accomplished by the addition of … a) a proton b) an electron c) a hydride ion d) a hydrogen atom Page 6 of 23

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29. Assume that an enzyme exists that would catalyze the following reaction: Malate + Acetaldehyde

Oxaloacetate + Ethanol

Given the standard reduction potentials for the half reactions are: Oxaloacetate + 2e + 2H+ Acetaldehyde + 2e + 2 H+

Malate

E0′ = -0.17 V

Ethanol

E0′ = -0.20 V

in the standard state: a) ethanol will reduce oxaloacetate with a ΔE0′ of -0.03 V b) ethanol will reduce oxaloacetate with a ΔE0′ of +0.03 V c) malate will reduce acetaldehyde with a ΔE0′ of -0.03 V d) malate will reduce acetaldehyde with a ΔE0′ of +0.03 V 30. When a muscle is stimulated to contract aerobically, less lactic acid is formed than when it contracts anaerobically because: a) glycolysis does not occur to a significant extent under aerobic conditions b) muscle is metabolically less active under aerobic than anaerobic conditions c) the lactic acid generated is rapidly incorporated into lipids under aerobic conditions d) under aerobic conditions most of the pyruvate generated as a result of glycolysis is oxidized by the citric acid cycle rather than reduced to lactate 31. The structure shown below serves as a cofactor in: O CH

C H2 C H C

10

CH2

3OH

C H3 CH3O

C H3

O

a) b) c) d)

Pyruvate dehydrogenase reaction β-oxidation reactions Electron transport chain reactions Phosphorylation reactions

32. The thiolase reaction: a) Uses CoA to cleave the bond between α and β carbons of fatty acids Page 7 of 23

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b) Requires NAD+ as a cofactor c) Requires FAD as a cofactor d) Activates fatty acids by converting them to fatty acyl CoA 33. The following statements refer to the enzyme catalyzed formation of citrate, in the citric acid cycle. Which statement is incorrect? a) NADH is a product b) Acetyl CoA is a substrate c) Oxaloacetate is a substrate d) Citrate synthase is the catalyst 34. You are carrying out experiments with mitochondria in which you are measuring oxygen consumption with various substrates in the presence of different inhibitors. With one particular substrate you find that its oxidation is inhibited by cyanide and antimycin A, but not by rotenone. The substrate could have been, a) α-ketoglutarate b) Malate c) Pyruvate d) Succinate 35. The mechanism of rotation of the C10 ring of ATP synthase depends on the making and breaking of an ionic interaction (“salt bridge”) between two amino acids. These two amino acids are… a) lysine and glutamate b) arginine and aspartate c) lysine and asparagine d) arginine and glutamate 36. Which of the following enzymes does not catalyze an oxidative decarboxylation (oxidation plus the release of CO2)? a) Alpha ketoglutarate dehydrogenase b) Pyruvate dehydrogenase c) Malate dehydrogenase d) Isocitrate dehydrogenase 37. All of the enzymes of the Krebs cycle are soluble in the mitochondrial matrix, except for one which is located in the mitochondrial inner membrane. This one is: a) Succinate dehydrogenase b) Malate dehydrogenase c) α-ketoglutarate dehydrogenase d) Isocitrate dehydrogenase Page 8 of 23

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38. The P/O ratio for NADH, in standard aerobic mitochondrial respiration, is approximately: a) 1.5 b) 2.0 c) 2.5 d) 3.0 39. In the course of the Krebs cycle, the molecule shown on the right is: a) Converted to malate by the addition of water b) Reduced to malate by NADH c) Reduced to malate by FADH2 d) Condensed with acetyl CoA to form citrate 40. All of the following methods are useful in elucidating metabolic pathways except, a) The use of CNBr to cleave polypeptides at the c-terminal end of methionine residues b) The use of metabolic inhibitors c) The use of auxotrophic mutants d) The use of radioactively labeled substrates

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Short answer questions. Answer all questions. 40 marks total. 1. Amino acids (5 marks) From the clue provided, identify (by name) the corresponding “building block” amino acid. The first answer is given, as an example. (1 mark each = 5 marks total) Clue Indole ring i) ii)

Replacement of the S atom of cysteine by an O atom would give this amino acid The side chain (R) = -CH2-CH2-S-CH3

Answer tryptophan Serine Methionine

iii)

Can act as a general acid as well as a general base at physiological pH

Histidine

iv)

The R-group links the α-N to the α-C

Proline

v)

The side chain contains a positively charged amino group at physiological pH

Lysine

2. Charge on amino acid side chains (3 marks) 3-methylhistidine is a post-translationally modified form of histidine with a side chain pKa of 5.8. Calculate the net charge on 3-methylhistidine side chain at pH 6.3 (The ionization states of 3-methylhistidine are same as that of histidine). (3 marks) (Show all your work for full marks) Net charge = +0.24

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3. Aerobic energy generation (5 marks) i. What is the reaction that connects glycolysis to the citric acid cycle? (1 mark) The oxidative dehydrogenation of pyruvate to acetyl-CoA

ii.

Write a balanced equation for the above reaction (in words, structures not required). (2.5 marks)

Pyruvate + CoA + NAD+ -----------pyruvate dehydrogenase----- acetyl-CoA + CO2 + NADH + H+

iii. People with beriberi, a disease caused by thiamine (vitamin B1) deficiency, have elevated levels of blood pyruvate. How is this effect related to a deficiency of thiamine? (1.5 mark) Thiamine is an important cofactor for the enzyme pyruvate dehydrogenase, and in case of thiamine (vitamin B1) deficiency the enzyme effectiveness will be diminished, causing accumulation in the pyruvate in cell, as a result of not being able to convert that pyruvate to acetyl-CoA.

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4. The citric acid cycle (5 marks) i. What is the most important role played by the TCA cycle in terms of bioenergetics? (1 mark) The citric acid cycle plays a major role in making most of the reduced cofactors, (3 NADH and 1FADH2 per each cycle), that are required for ETC to function and generate most of the cell ATP.

ii. The citric acid cycle is frequently described as the major pathway of aerobic catabolism, which means that it is an oxygen-dependent degradative process. However, none of the reactions of the cycle directly involves oxygen as a reactant. Why is the pathway oxygendependent? (1 mark) Since the citric acid cycle must use the NAD+ and FAD, these cofactors have to be oxidized again after being reduced by the TCA. The only way to do so is by using these reduced cofactors (NADH and FADH2) in the ETC, and in doing so ETC require O2 to be available as a final electeon acceptor of these reduced factors. In summery, even though the TCA does not require O2 directly, it does require it indirectly by relaying on ETC to recycle (oxidize) its reduced cofactors.

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iii. In which reaction of the citric acid cycle does a substrate level phosphorylation occur? Name the enzyme and write a balanced equation for the reaction (in words, structures not required). (3 marks) Reaction: the substrate level phosphorylation of succinyl-CoA to succinate Enzyme: succinyl-CoA synthetase Equation: Succinyl-CoA + pi + GDP ---------succinyl-CoA synthetase--- succinate + CoA + GTP

5. Beta oxidation (6 marks) i. The energy required for the activation of fatty acids to fatty acyl-CoA is provided by the hydrolysis of ATP to AMP + PPi. Explain why this mode of ATP hydrolysis leads to the release of twice the amount of energy as the hydrolysis of ATP to ADP + Pi? (2 marks) When ATP is hydrolysed to ADP and Pi, only one phospho-anhydride bond is hydrolysed. One the other hand, when ATP is hydrolysed to AMP + ppi, the pyrophosphate (ppi) will further hydrolyse using the pyrophosphatase to make 2pi. In total the ATP to AMP reaction will yield a break of two phosphoanhydride bonds (2ATP energy) ATP ---- AMP + ppi (1 bond E) Ppi --- 2pi (1 bond E) In total: ATP ---- AMP + 2pi (2 phospho-anhydride bonds E)

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ii. Indicate the number of acetyl CoA, FADH2 and NADH that would be generated after completion of beta oxidation of a 16 carbon saturated fatty acid? (3 marks) # of acetyl CoA = ______8_______ # of FADH2 # of NADH

= ______7_______ = ____7_________

iii. What is the fate of the acetyl CoA produced in beta oxidation? (1 mark)

the acyl-CoA will be joined to citric acid cycle and further oxidized to make the reduce factors and GTP of citric acid cycle

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6. Oxidative phosphorylation (4 marks) The graph on the right was derived from an experiment carried out with isolated mitochondria suspended in a buffered medium. The addition of the indicated substances to the medium at the time points shown, leads to changes in O2 consumption (top line) and ATP synthesis (bottom line).

i.

Cyanide (CN-) is an inhibitor of the electron transport chain (ETC). However, the addition of CN- to the mitochondria not only inhibits the ETC but also inhibits ATP synthesis (bottom line on the graph). Explain the reason for this. (1 mark) When CN- is added, it will lead to the stop of electron flow and O2 consumption. This will cause the H+ pumps to stop, thereby destroying the electrochemical gradient that is essential for the H+ (protons) to go through ATP synthase. Therefore, with no H+ flowing to the ATP synthase ATP synthesis will be inhibited.

ii.

According to the chemio...