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UNIVERSITY OF EAST ANGLIA School of Biological Sciences Main Series UG Examination 2015-2016 MOLECULAR ENZYMOLOGY IN BIOLOGY AND MEDICINE BIO-6001A/ BIO-3C01 Time allowed: 3 hours Answer ALL questions in Section A and ONE question from Section B. Write answers to EACH SECTION in a SEPARATE booklet. The maximum number of marks available for your answers in SECTION A is 50 marks The maximum number of marks available for your answer in SECTION B is 50 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-6001A/ BIO-3C01 Copyright of the University of East Anglia Module Contact: Dr Andrew Hemmings, BIO Version 1 2 SECTION A: SHORT ANSWER QUESTIONS Answer ALL questions 1. How should the terms (i) R-factor and (ii) resolution be interpreted when used to describe the X-ray crystal structure of an enzyme? [5 marks] 2. Discuss briefly four considerations important in the rational design of inhibitors of a specific protease based on the sequence of its known peptide substrate. [4 marks] 3. You have a plasmid which has been generated for heterologous overexpression of a gene for an enzyme of interest, in Escherichia coli. State the steps involved in a method to introduce a single amino acid substitution in the enzyme active site. [5 marks] 4. Thrombin cleaves the tri-peptide substrate Gly-Val-Arg-pNA (corresponding to the cleavage sequence of its natural substrate, fibrinogen) very poorly. However, it cleaves the substrate Phe-Pro-Arg-pNA very efficiently. How do you account for this apparent discrepancy? [3 marks] 5. Some protein inhibitors of serine proteases are proteolytically cleaved by the enzyme, but others are not. Give three reasons that help to account for this difference. [3 marks] 6. Many serine proteases are modular proteins. Give two examples of the functional roles that the non-catalytic domains of these enzymes can play. [4 marks] 7. What are the two molecules that are commonly used as co-factors during the reactions catalysed by nucleic acid ligases? What is the chemical group contained within both molecules? [3 marks] 8. Briefly describe three important features of assays that have been most widely used to detect the biochemical activity of enzymes that break or join phosphodiester bonds in the backbone of DNA. [3 marks] 9. What features of DNA topoisomerases make them good targets for chemotherapeutic agents? Give one example of an antibiotic and one of an anticancer agent that targets a topoisomerase, in each case stating which enzyme they target. [5 marks] Section A continues on next page/ . . . BIO-6001A/ BIO-3C01 Version 1 3 Section A continued. . . 10. Type I DNA topoisomerases are sub-divided into two main sub-types. What are the principle mechanistic differences between these two sub-types? [4 marks] 11. Name four different oxygen species that aerobic cells can be exposed to. [4 marks] 12. Describe the three metal cofactors required by nitrogenase for nitrogen fixation. [3 marks] 13. While studying the kinetics of oxidation of substrates X and Y catalysed by Cytochrome P450 3A5 you generate the following graphs. Explain what could be happening in each case. [4 marks] Substrate Y Substrate X 80 70 50 Ac vity Ac vity 60 40 30 20 10 0 0 100 200 300 18 16 14 12 10 8 6 4 2 0 0 100 200 300 [Y] mM [X] mM END OF SECTION A START YOUR ANSWER TO THE NEXT SECTION IN A NEW BOOKLET Section B begins on next page/ . . . TURN OVER BIO-6001A/ BIO-3C01 Version 1 4 SECTION B: ESSAY QUESTION Answer ONE question [50 marks] 14. Explain the strategies commonly employed by enzymes to accelerate the rates of biochemical reactions. Illustrate your answer by reference to relevant examples. 15. Using your knowledge of the catalytic mechanism of serine proteases, and the key features that contribute to it, explain how the activity of these enzymes is regulated biologically and how it can be manipulated therapeutically. 16. DNA helicases and DNA topoisomerases perform key roles in DNA replication. Explain what these roles are and how these enzymes carry them out. What is the role of ATP in the reactions of helicases and topoisomerases? END OF PAPER BIO-6001A/ BIO-3C01 Version 1