Student Number: THE UNIVERSITY OF MANITOBA April 10, 2000, 9:00 AM - 12:00 PM Page 1 (of 4) Biochemistry II Lab Section Final Examination

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1 Name: Student Number: THE UNIVERSITY OF MANITOBA April 10, 2000, 9:00 AM - 12:00 PM Page 1 (of 4) Biochemistry II Lab Section Final Examination Examiner: Dr. A. Scoot 1. Answer ALL questions.. 2. Questions must be answered in the space provided 3. The last page provides space for preliminary work. 1. Chromatographic methods are some of the most widely used techniques in biochemistry for separation and identification of components in a mixture. Name three of the four chromatographic methods used in the lab this term and include a brief explanation of how each method works

2 April 10, 2000, 9:00 AM - 12:00 PM Page 2 (of 4) Biochemistry II Lab Section Final Examination Examiner: Dr. A. Scoot 3 2. You are given a solution containing an enzyme that converts B into A. Outline briefly what you would do to determine the specific activity of this enzyme Answer ONE (1) of the following two parts, a or b. Explain the significance and mode of action of one of the two following sets of reagents in relation to their use in this term s biochemistry laboratories. (a) (b) Barium hydroxide and arsenomolybdate solutions (used in same experiment) Perchloric acid and potassium hydroxide (used in same experiment)

3 April 10, 2000, 9:00 AM - 12:00 PM Page 3 (of 4) Biochemistry II Lab Section Final Examination Examiner: Dr. A. Scoot Two different columns were used to see which gave the better separation of lysozyme from a filtered buffered egg white preparation (FBEW). The lysozyme activity in 0.1 ml of a l in 2 dilution of FBEW was found to be 1.50 enzyme units. The protein content of a 1 in 20 dilution of the FBEW was found by biuret assay to be 3.0 mg/ml. A 5.0 ml sample of FBEW was applied to column A and another 5.0 ml applied to column B. Several 10.0 ml fractions were collected from each column. For column A the enzyme activity was found in fraction 2 where 0.20 ml was found to contain 1.50 enzyme units of lysozyme and the protein content of a 1 in 5 dilution was found to be 3.00 mg/ml. For column B the enzyme activity was found in fraction 5 where 0.20 ml was found to contain 1.50 enzyme units of lysozyme and the protein content of a 1 in 2 dilution was found to be 3.75 mg/ml. (a) Calculate the percentage of the total protein and of the total enzyme activity recovered in column A, fraction 2 and in column B, fraction 5. (b) Which of the two columns gave the greatest degree of purification? Show calculations and cite values to support your answer. Question #4 continued on next page

4 April 10, 2000, 9:00 AM - 12:00 PM 4 (of 4) Biochemistry II Lab Section Final Examination Examiner: Dr. A. Scoot (c) Briefly explain the principle behind the assay for lysozyme. Working Space - (Not to be marked)

5 Name: Student Number: THE UNIVERSITY OF MANITOBA April 10, 2000, 9:00 AM - 12:00 PM Page 1 (of 8) 1. Answer ALL questions.. 2. Questions must be answered in spaces provided. 3. The last page provides space for preliminary work Using structural formulae, and the names of enzymes and coenzymes, describe the pathway by which both nitrogen atoms in urea can be derived from alanine.

6 April 10, 2000, 9:00 AM - 12:00 PM Page 2 (of 8) 8 2. Describe a plausible series of reactions by which an organism provided with aspartic acid as its sole source of carbon can synthesize glucose. Use compound, enzyme, and coenzyme names in your answer. What is the net consumption or generation of ATP equivalents during this process? Be sure to show how you arrived at your answer Write the names and structures of at least the business ends of two co-enzymes that are carriers of single carbon compounds.

7 April 10, 2000, 9:00 AM - 12:00 PM Page 3 (of 8) 8 4. Using structural formulae and the business ends of the co-enzymes write the mechanism for the reaction catalysed by the a-ketoglutarate dehydrogenase complex. Comment on the free energy change for this reaction.

8 April 10, 2000, 9:00 AM - 12:00 PM Page 4 (of 8) 5 5. Using compound names, write the TCA Cycle pathway. (Enzyme and co-enzyme names are not required.) Indicate those amino acids which are catabolized to TCA cycle intermediates and identify their points of entry into the Cycle. Name the three other amino acid oxidation products that are not TCA cycle intermediates With the aid of a diagram, briefly describe how 3', 5'cyclic AMP functions in the regulation of glycogen metabolism in muscle cells.

9 April 10, 2000, 9:00 AM - 12:00 PM Page 5 (of 8) 3 7. Draw the structures of two base pairs, (a) one involving guanine and cytosine rings and (b) the second involving guanine and uracil rings showing the location of hydrogen bonds. (a) (b) 7 8. Using the Z-scheme as the basis of your answer, show how both ATP and NADPH are generated by the light reactions of photosynthesis. Indicate how many (a) photons are required, (b) ATPs are produced and (c) oxygen molecules (O 2 ) are produced when one molecule of NADP + is reduced by this scheme.

10 April 10, 2000, 9:00 AM - 12:00 PM Page 6 (of 8) 9 9. Using structures and compound, enzyme and coenzyme names how two molecules of acetate would be converted into one molecule of butyryl CoA. Indicate clearly why citrate and CO 2 are required for this process

11 April 10, 2000, 9:00 AM - 12:00 PM Page 7 (of 8) Using diagrams, explain the molecular mechanisms of repression and activation that operate in the lac operon Using diagrams, outline the mechanism for the initiation of translation.

12 April 10, 2000, 9:00 AM - 12:00 PM Page 8 (of 8) Using structures, and compound, enzyme and co-enzyme names, describe a pathway by which acetate can be converted into the amino acid alanine by a bacterium.

13 April 10, 2000, 9:00 AM - 12:00 PM Working Page Biochemistry I Lecture Section Final Examination Working Space - (Not to be Marked)