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UNIVERSITY OF EAST ANGLIA School of Biological Sciences Main Series UG Examination 2012-2013 BIOCHEMISTRY BIO-2B01 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 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. Notes are not permitted in this examination. Do not turn over until you are told to do so by the Invigilator. BIO-2B01 Module Contact: Dr Richard Bowater, BIO Copyright of the University of East Anglia Version 1 2 SECTION A: MULTIPLE CHOICE AND SHORT ANSWER QUESTIONS Answer ALL questions. Unless stated otherwise all multiple choice questions have ONE answer. 1. Erythrose 4-phosphate is a precursor of: a) b) c) d) e) [1 mark] Aspartic acid Cysteine Phenylalanine Serine Threonine 2. An important intermediate in the biosynthetic pathway to aromatic amino acids is: [1 mark] a) Benzoic acid b) Lactate c) Orotate d) Shikimate e) α-ketoglutarate 3. 5-Phosphoribosyl-1-pyrophosphate (PRPP) is a synthetic precursor for all of the following except: [1 mark] a) b) c) d) e) 4. AMP Arginine Histidine Tryptophan UMP Precursors for the biosynthesis of the pyrimidine ring system include: [1 mark] a) b) c) d) e) Carbamoyl phosphate and aspartate Glutamate, NH3, and CO2 Glycine and succinyl-CoA Glycine, glutamine, CO2, and aspartate Inosine and aspartate Section A continues/ . . . BIO-2B01 Version 1 3 . . . /Section A continued 5. Which one of the following types of mechanisms does not play a role in the reversible alteration of enzyme activity? [1 mark] a) b) c) d) e) Activation by cleavage of an inactive zymogen Allosteric response to a regulatory molecule Alteration of the synthesis or degradation rate of an enzyme Covalent modification of the enzyme Interactions between catalytic and regulatory subunits 6. Which one of the following conditions is not caused by a dietary deficiency of a vitamin? [1 mark] a) b) c) d) e) Werner-Korsakoff Syndrome Pellagra Phenylketonuria Beri-Beri Rickets 7. Which of the following best describes the function of nicotinamide adenine dinucleotide? [1 mark] a) b) c) d) e) A 1 electron 1 proton carrier A 1 electron 2 proton carrier A 2 electron 1 proton carrier A 4 electron 2 proton carrier A 2 electron 2 proton carrier 8. Which one of the following glucose transporters is constitutively expressed in the membranes of pancreatic beta cells? [1 mark] a) b) c) d) e) GLUT1 GLUT2 GLUT3 GLUT4 GLUT5 Section A continues/ . . . TURN OVER BIO-2B01 Version 1 4 . . . /Section A continued 9. Which of the following statements is true? [1 mark] a) Fatty acid biosynthesis takes place in the cytosol, uses NADH as a cofactor and requires the participation of the 3-carbon intermediate propionyl-CoA b) Fatty acid biosynthesis takes place in the mitochondria, uses NADPH as a cofactor and requires the participation of the 3-carbon intermediate propionyl-CoA c) Fatty acid biosynthesis takes place in the cytosol, uses NADH as a cofactor and requires the participation of the 3-carbon intermediate malonyl-CoA d) Fatty acid biosynthesis takes place in the mitochondria, uses NADPH as a cofactor and requires the participation of the 3-carbon intermediate malonyl-CoA e) Fatty acid biosynthesis takes place in the cytosol, uses NADPH as a cofactor and requires the participation of the 3-carbon intermediate malonyl-CoA 10. In Fatty Acid Synthase Type I, Malonyl CoA is esterified to the SH group of which one of the following? [1 mark] a) b) c) d) e) Cysteine 4’-Phosphopantetheine Coenzyme A Lipoic Acid Glutathione 11. The type of membrane transport that uses ion gradients as the energy source is: [1 mark] a) b) c) d) e) Facilitated diffusion Passive transport Primary active transport Secondary active transport Simple diffusion Section A continues/ . . . BIO-2B01 Version 1 5 . . . /Section A continued 12. Oligomycin and 2,4-dinitrophenol (DNP) are inhibitors of mitochondrial aerobic phosphorylation. Which of the following statements correctly describes the mode of action of these two inhibitors? [1 mark] a) 2,4-dinitrophenol inhibits the respiratory chain, and oligomycin inhibits the synthesis of ATP b) Oligomycin and 2,4-dinitrophenol inhibit the synthesis of ATP c) Oligomycin, and 2,4-dinitrophenol compete with O2 for cytochrome oxidase (Complex IV) d) Oligomycin inhibits synthesis of ATP and 2,4-dinitrophenol inhibits the respiratory chain e) Oligomycin inhibits the respiratory chain, whereas 2,4-dinitrophenol prevents the synthesis of ATP 13. 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) It 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 14. During oxidative phosphorylation, the proton motive force that is generated by electron transport is used to: [1 mark] a) b) c) d) e) Create a pore in the inner mitochondrial membrane Generate the substrates (ADP and Pi) for the ATP synthase Induce a conformational change in the ATP synthase + Oxidize NADH to NAD Reduce O2 to H2O Section A continues/ . . . TURN OVER BIO-2B01 Version 1 6 . . . /Section A continued 15. Which of the following is not involved in the specificity of signal transduction? [1 mark] a) b) c) d) e) 16. Scatchard analysis can provide information on: a) b) c) d) e) 17. [1 mark] Enzyme cascades Enzyme mechanisms Gated ion channels Protein phosphorylation Receptor-ligand interactions Which of the following does not involve cyclic AMP? a) b) c) d) e) 18. 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 [1 mark] Regulation of glycogen synthesis and breakdown Regulation of glycolysis Signalling by acetylcholine Signalling by epinephrine Signalling by glucagon How is trypsinogen converted to trypsin? [1 mark] a) A protein kinase-catalyzed phosphorylation event converts trypsinogen to trypsin b) Binding of Ca2+ to trypsinogen converts it to trypsin c) Proteolysis of trypsinogen forms trypsin d) Trypsinogen dimers bind an allosteric modulator, cAMP, causing dissociation into active trypsin monomers e) Two inactive trypsinogen dimers pair to form an active trypsin tetramer Section A continues/ . . . BIO-2B01 Version 1 7 . . . /Section A continued 19. 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) Warfarin Vitamin K Calcium chloride -carboxy glutamic acid Oxygen 20. Which of the following describes the overall conformation of a protein, taking account of the organisation of multiple polypeptide chains, including any ligands such as cofactors and metal ions? [1 mark] a) b) c) d) e) The primary structure of a protein The secondary structure of a protein The tertiary structure of a protein The quaternary structure of a protein The gene sequence associated with a protein 21. From the following seven options, select the option(s) that best fit the biochemical descriptions of cytochromes P450 that are outlined below. Each option may be used once, more than once, or not at all. [5 marks] List of seven options: a) b) c) d) e) f) g) CYP1A1 CYP2A1 CYP1A3 CYP2C9 CYP2D6 Class I Class II Biochemical descriptions of cytochromes P450: i) Which cytochromes P450 receive electrons from ferredoxins? ii) Which cytochromes P450 share > 55% sequence identity? iii) Which cytochrome P450 is found in the liver and processes several commonly used drugs including warfarin? iv) Which cytochrome P450 is present in high levels in many persons of Ethiopian and Saudi Arabian origin? Section A continues/ . . . TURN OVER BIO-2B01 Version 1 8 . . . /Section A continued 22. Describe how to prepare 250 ml of 0.2 M phosphate buffer pH 6.8, containing 0.2 M KCl. Note that to obtain phosphate buffer at pH 6.8, the molar ratio of KH2PO4 to Na2HPO4 should be 1.15:1.00. The relevant molecular masses are given in Table 1 below. [5 marks] Chemical KCl Na2HPO4 KH2PO4 Table 1 Molar mass (g.mol-1) 74.55 142.0 136.1 23. Fill in the missing words ((a)-(e)) in the following statements. Note that the answer to part (c) is a number. (Write your answers in your answer booklet, do not write your answers on the exam paper.) [5 marks] ‘(a)___________ enzymes catalyse the (b)________ cleavage of peptide bonds. Catalysis by serine proteases is essentially a (c)_______-step process and proceeds via a (d)_________ intermediate known as the (e)_________ complex.’ 24. 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 Section A Q24 continues/… BIO-2B01 Version 1 9 …/Section A Q24 continued 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) Trypsin has a “primary specificity” that relates to the type of peptide bonds that it cleaves. What is the primary specificity of trypsin? [1 mark] d) Name a related serine protease that has different primary specificity. Include details of its primary specificity. [2 marks] END OF SECTION A Section B begins on next page/ . . . TURN OVER BIO-2B01 Version 1 10 SECTION B: Answer ALL PARTS of the question 25. A sample of lactate dehydrogenase (LDH) has been purified. Throughout the purification protocol the specific activity of LDH in the sample was determined using an assay involving NADH, which has an extinction coefficient at 340 nm of 6.22 x 103 M-1.cm-1. (Note that NAD+ has an extinction coefficient of zero at 340nm.) The following data refer to experiments that used cuvettes of 1 cm path length. a) What reaction is catalysed by LDH? In your answer, include details about any cofactors that are required during the reaction. [4 marks] b) As the sample became more pure in terms of its LDH content, what would you expect to happen to its specific activity for LDH? Briefly, explain your answer. [4 marks] c) The assays of LDH activity were set up as a 4.0 ml solution with an initial absorbance at 340 nm of 0.95. How much NADH (in moles) was present in this sample? [6 marks] d) For the most pure sample of LDH, 0.01 ml of the sample was found to contain 12 micrograms of protein. Calculate the concentration of protein in the sample in mg.ml-1. [3 marks] e) Assuming the sample referred to in part (d) is composed entirely of LDH and that the relative molar mass (Mr) of LDH is 35,000, what is the concentration of the sample analysed in part (d)? Give your answer in M to 2 decimal places. [3 marks] f) For the sample referred to in part (d), the LDH activity assay produced an absorbance change at 340 nm of 0.52 per minute when 0.05ml of sample was added to the reaction volume of 4.0 ml. For this sample, what was the specific activity of LDH (in μmol NADH min-1 .mg protein-1)? [8 marks] g) If the LDH assays were repeated in the presence of oxamate, what would you expect to happen to the rate of LDH activity? Briefly, justify your answer. [2 marks] END OF SECTION B Section C begins on next page/ . . . BIO-2B01 Version 1 11 SECTION C: ESSAY QUESTION Answer ONE question [30 marks] 26. The selective translocation of small metal ions across biological membranes is achieved through the use of membrane proteins. Using appropriate examples, compare and contrast how the transport of metal ions across membranes is achieved by membrane proteins that use passive diffusion or active transport mechanisms. 27. Ribonucleotide reductase from Escherichia coli converts ribonucleotides to their deoxy form. Describe the various biochemical reactions that are performed by this enzyme, making sure that you provide a specific description of the ways in which its various polypeptides are arranged. What allosteric regulatory reactions influence the activity of this enzyme? 28. Compare and contrast the mechanisms by which animal cells respond to growth factors and adrenaline. In your answer include details about the mechanism of interaction between these ligands and the cell surface receptors. END OF PAPER BIO-2B01 Version 1 12 BIO-2B01 EXAMINATION MARKERS 2012-13 [Do not print this when printing to take to Examinations Office! This is for our information only.] Question No. Section A Section B Section C Q26 Section C Q27 Section C Q28 BIO-2B01 1st Marker Prof J Butt Dr R Bowater Dr T Clarke Dr R Bowater Dr Charles Brearley 2nd Marker Prof V Ellis Dr Charles Brearley Dr N Watmough Dr N Watmough Dr T Clarke Version 1