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UNIVERSITY OF EAST ANGLIA School of Biological Sciences Main Series UG Examination 2015-2016 BIOCHEMISTRY BIO-5002A Time allowed: 2 hours Answer ALL questions in Section A, ALL PARTS of the question in Section B and ONE question from Section C. Write answers to EACH SECTION in the Answer Grid or a SEPARATE booklet. The maximum number of marks available for your answers in SECTION A is 40 marks The maximum number of marks available for your answer in SECTION B is 30 marks The maximum number of marks available for your answer in SECTION C is 30 marks The TOTAL number of marks available for the paper is 100 Numbers in square brackets [ ] indicate the relevant mark applied to each part of the question. Graph paper is provided. Notes are not permitted in this examination. Do not turn over until you are told to do so by the Invigilator. BIO-5002A Copyright of the University of East Anglia Module Contact: Dr Richard Bowater, BIO Version 1 2 SECTION A: MULTIPLE CHOICE AND SHORT ANSWER QUESTIONS Answer ALL questions. Answer multiple choice questions in the answer grid provided and attach this to your booklet for Section A. 1. The synthesis of purine and pyrimidine nucleotides differ in that: [1 mark] a) ATP is required in the synthesis of purines but not in the synthesis of pyrimidines b) purine biosynthesis starts with the formation of PRPP (phosphoribosyl pyrophosphate), whereas pyrimidines incorporate PRPP near the end of the pathway c) pyrimidine formation requires a tetrahydrofolate (THF) derivative, whereas purine formation does not d) pyrimidine biosynthesis is tightly regulated in the cell, whereas purine biosynthesis is not e) pyrimidines go through many steps, adding a single carbon or nitrogen each time, whereas the basic skeleton for purines is formed from two main precursors 2. An important intermediate in the biosynthetic pathway to aromatic amino acids is: [1 mark] a) b) c) d) e) 3. One amino acid directly involved in the purine biosynthetic pathway is: a) b) c) d) e) 4. benzoic acid lactate orotate shikimate α-ketoglutarate (2-oxo-glutarate) [1 mark] alanine aspartate glutamate leucine tryptophan Which of the following molecules is not an inhibitor of glutamine synthetase? [1 mark] a) b) c) d) e) AMP valine histidine glycine glucosamine-6-phosphate Section A continues on next page/ . . . BIO-5002A Version 1 3 Section A continued. . . 5. In which of the following enzymes would you not expect to find a domain known as a "Rossmann fold"? [1 mark] a) b) c) d) e) 6. Which of the following enzymes is not part of the fatty acid synthase found in the cytosol of mammalian cells? [1 mark] a) b) c) d) e) 7. Alcohol Dehydrogenase Acetyl-CoA Carboxylase Acetaldehyde Dehydrogenase Carbonyl Oxidoreductase Lactate Dehydrogenase Ketoacyl synthase Enoyl CoA isomerase Malonylacetyl transferase Thioesterase Dehydratase Which of these statements about facilitated diffusion across a membrane is true? [1 mark] a) Facilitated diffusion involves a specific membrane protein that lowers the activation energy for movement of the solute through the membrane b) Facilitated diffusion can increase the magnitude of a transmembrane concentration gradient of the diffusing solute c) Facilitated diffusion is dependent upon the solubility of the transported solute in the nonpolar interior of the lipid bilayer d) Facilitated diffusion is responsible for the transport of gases, such as O2, N2, and CH4, across biological membranes e) The rate of solute transport never reaches saturation 8. Upon the addition of 2,4-dinitrophenol (DNP) to a suspension of mitochondria carrying out oxidative phosphorylation linked to the oxidation of malate, all of the following occur except: [1 mark] a) b) c) d) e) the rate of ATP production increases oxygen consumption increases the P/O ratio drops from a value of approximately 2.5 to zero the proton gradient dissipates the rate of transport of electrons from NADH to O2 becomes maximal Section A continues on next page/ . . . TURN OVER BIO-5002A Version 1 4 Section A continued. . . 9. Which of the following statements about the chemiosmotic theory is correct? [1 mark] a) Electron transfer in mitochondria is accompanied by an asymmetric release of protons on one side of the inner mitochondrial membrane b) The chemiosmotic theory predicts that oxidative phosphorylation can occur even in the absence of an intact inner mitochondrial membrane c) The effect of uncoupling reagents is a consequence of their ability to carry electrons through membranes d) The membrane ATP synthase has no significant role in the chemiosmotic theory e) For each proton transported out of the mitochondria, one electron must be transferred along the electron transfer chain 10. Which of the following statements is not true about cytochrome P450 enzymes? [1 mark] a) b) c) d) The active site contains magnesium They belong to a general class of enzymes called monooxygenases There are over 30 different cytochrome P450 enzymes Variation in the cytochrome P450 enzyme profile between individuals can explain individual variation in drug susceptibility e) Human cytochrome P450 enzymes are bound to membranes 11. Which of the following is not involved in the specificity of signal transduction? [1 mark] a) b) c) d) e) 12. Interactions between receptor and signal molecules Location of receptor molecules Structure of receptor molecules Structure of signal molecules Transmembrane transport of signal molecules by receptor molecules Scatchard analysis can provide information on: a) b) c) d) e) [1 mark] enzyme cascades enzyme mechanisms gated ion channels protein phosphorylation receptor-ligand interactions Section A continues on next page/ . . . BIO-5002A Version 1 5 Section A continued. . . 13. Which of the following does not involve cyclic AMP? a) b) c) d) e) 14. Regulation of glycogen synthesis and breakdown Regulation of glycolysis Signalling by acetylcholine Signalling by epinephrine (adrenaline) Signalling by glucagon A patient has been poisoned by a rodenticide of the coumarin-type. Which of the following is likely to be an effective treatment? [1 mark] a) b) c) d) e) 15. [1 mark] Warfarin Vitamin K Calcium chloride γ-carboxy glutamic acid Oxygen Ubiquitin is a: a) b) c) d) e) [1 mark] component of the electron transport system protease protein kinase protein phosphorylase protein that tags another protein for proteolysis For all remaining questions in Section A, please use an answer booklet. Remember to attach your multiple choice answer grid to the booklet. Section A continues on next page/ . . . TURN OVER BIO-5002A Version 1 6 Section A continued. . . For all remaining questions in Section A, please use an answer booklet. Remember to attach your multiple choice answer grid to the booklet. 16. Answer ALL parts (a), (b), (c) and (d). Trypsin is an example of a hydrolase enzyme. The x-ray structure of purified trypsin has been solved and the position of atoms present within the active site is as follows: A B C The “catalytic triad” that is present in all serine proteases consists of 3 specific, but different, types of amino acids. The catalytic triad for trypsin is highlighted above, with the 3 amino acids designated as “A”, “B” and “C”. Use the information above and the image to answer the following questions. (a) What general types of reactions are catalysed by “Hydrolases”? [1 mark] (b) For the 3 amino acids highlighted in the image above, which of them is the catalytic serine? (Answer “A”, “B” or “C”.) [1 mark] (c) Serine proteases have a “primary specificity” that relate to the “P1” residue of the substrate. What is the primary specificity of trypsin? [1 mark] (d) What two key features of the primary specificity pocket (S1) of serine proteases determine which P1 residue can be bound? [2 marks] Section A continues on next page/ . . . BIO-5002A Version 1 7 Section A continued. . . 17. Name the five different biochemical classes of amino acids and name an example of each class. [5 marks] 18. With the help of a diagram, name and highlight the arrangements of the five mitochondrial transmembrane proteins that are involved in the respiratory transport chain and ATP synthesis. Within your diagram, show how the protons move across the inner mitochondrial membrane. [5 marks] 19. For the following options, match the enzyme to its preferred substrate. Only one answer should be provided for each enzyme and each answer should be listed no more than once. [5 marks] List of five enzymes: a) Ribonucleotide reductase b) Aspartate transcarbamoylase c) Glutamine synthetase d) Hexokinase e) Pyruvate kinase List of biological molecules: i) Glucose ii) dATP iii) Lactose iv) Glutamate v) ADP vi) Phosphoenolpyruvate vii) Tryptophan viii) 5-phosphoribosyl-1-pyrophosphate (PRPP) ix) Aspartate x) Phenylalanine 20. Answer ALL parts (a), (b) and (c). A sample of Enzyme X has been purified and biochemical studies were conducted on it. (a) During the purification process, as the sample became more pure in terms of its content of Enzyme X, what would you expect to have happened to the specific activity for this enzyme? Briefly, explain your answer. [2 marks] Section A Q20 continues on next page/ . . . TURN OVER BIO-5002A Version 1 8 Section A Q20 continued. . . (b) For the purified Enzyme X, optimal activity of the enzyme was shown to require the presence of pyruvate and NADH. What protein is likely to constitute Enzyme X? [1 mark] (c) If the assays were repeated in the presence of oxamate in addition to pyruvate, what would you expect to happen to the rate of activity of Enzyme X? Briefly, justify your answer. [2 marks] END OF SECTION A START YOUR ANSWER TO THE NEXT SECTION IN A NEW BOOKLET SECTION B: DATA HANDLING QUESTION Answer ALL PARTS of the question [30 marks] 21. (a) A series of protein samples were prepared, with an aim to use the Bradford assay to determine the concentration of protein in each. In one sentence, describe the principles behind the Bradford assay. In addition, suggest one alternative approach that could be used to determine the concentration of protein in each sample. [4 marks] (b) The concentration of protein in some experimental samples was measured using the Bradford assay. A buffer was provided for the experiment, but KCl had to be added to be at a final concentration of 0.2 M. How much solid KCl was added to prepare 300 ml of buffer containing 0.2 M KCl? Note that the molar mass of KCl [2 marks] is 74.55 g.mol-1. (c) Using a solution of bovine serum albumin (BSA) at a known concentration, a calibration curve for a Bradford assay was prepared. Different volumes of BSA were mixed with water so that the total volume of protein solution in each measurement was 0.1 ml. Table 1 (on the next page) shows the absorbance readings at 595 nm (A595) for the various amounts of protein. On the graph paper provided, plot the amount of protein in each sample against its absorbance at 595 nm. Comment briefly on the relationship between the protein concentration and the measured absorbance as revealed by your plot. [6 marks] Section B continues on next page/ . . . BIO-5002A Version 1 9 Section B continued. . . Amount of BSA (microgram) Absorbance at 595 nm 0 0.000 5 0.280 10 0.590 15 0.847 20 1.132 25 1.428 30 1.595 40 1.830 50 1.915 Table 1. Bradford assay analysis of samples containing different amounts of BSA. (d) A solution of Protein A at an unknown concentration was analysed using the same Bradford assay protocol. For 0.1 ml of Protein A, the A595 was 0.520. Using the graph prepared in part (c) as your calibration curve, calculate the [6 marks] concentration (in mg.ml-1) of protein A. (e) Assuming the relative molecular mass (Mr) of Protein A is 30,345, what is the molar concentration of the sample analysed in part (d)? Give your answer in µM to 2 decimal places. [4 marks] (f) A solution of Protein B at an unknown concentration was analysed using the same Bradford assay protocol. For a mixture of 0.05 ml Protein B plus 0.05 ml of water, the A595 was 1.195. Using the graph prepared in part (c) as your calibration curve, calculate the concentration (in mg.ml-1) of Protein B. [4 marks] (g) If the Bradford assay was performed on an undiluted sample of Protein B, would the A595 measurement be higher or lower compared to that reported in part (f)? Would the analysis of diluted or non-diluted Protein B provide a more accurate measurement of the concentration of protein in the sample? Explain your answer. [4 marks] END OF SECTION B START YOUR ANSWER TO THE NEXT SECTION IN A NEW BOOKLET Section C begins on next page/ . . . TURN OVER BIO-5002A Version 1 10 SECTION C: ESSAY QUESTION Answer ONE question [30 marks] 22. The selective translocation of small metal ions across biological membranes is achieved through the use of membrane proteins by either facilitated diffusion or active transport. Describe, using one appropriate example of each mechanism, how the selective membrane transport of sodium and potassium is achieved. 23. Some metabolic pathways are fundamental to the growth and function of all cells. For such central aspects of metabolism it is essential that specific molecules are maintained at appropriate concentrations. By referring to specific types of biomolecules that are fundamental to cell growth and function, discuss the strategies that have evolved to allow regulation of metabolic pathways. Your answer should include one example that highlights how a metabolic pathway is activated and one example that highlights how a metabolic pathway is inhibited. 24. What is the relevance of the equilibrium binding constant Kd to cell signalling initiated by binding to cell surface receptors of ligands, such as growth factors or adrenaline? Within your answer describe how binding of a named ligand mediates the response of the cell. END OF PAPER BIO-5002A Version 1 UNIVERSITY OF EAST ANGLIA School of Biological Sciences Main Series UG Examination 2015-2016 BIOCHEMISTRY BIO-5002A Registration Number SECTON A: Answer Grid (for Multiple Choice Questions only) Place a single cross in the appropriate box Question No. BIO-5002A A B C D E Marks given Marks available 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 1 10 1 11 1 12 1 13 1 14 1 15 1 Version 1