EXPERIMENT ONE. Answer the following sections: A) Name the compound added to RNAse A to break the disulphide bonds. (5 marks)

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1 (100 marks = 33%) 2. Christian Anfinsen performed significant experiments on the enzyme Ribonuclease A (RNAse A) that led to his gaining the Nobel Prize in Chemistry in RNAse A has 124 residues in its Primary Structure and has 4 disulphide bonds. EXPERIMENT ONE. Answer the following sections: A) Name the compound added to RNAse A to break the disulphide bonds. (5 marks) B) Name the second compound that Anfinsen added to RNAse A. (5 marks) C) Indicate TRUE or FALSE. This second compound when added to RNAse A: (5 marks) i) Broke the remaining disulphide bonds ii) Disrupted Van de Waals contacts in the enzyme iii) Induced salting out of the protein iv) Broke ionic bonds in the protein v) Broke hydrogen bonds in RNAse A D) Indicate TRUE or FALSE. After addition of these two compounds to RNAse A (5 marks) i) it dehydrated ii) it oxidised

2 iii) it precipitated iv) it denatured v) it condensed E) What was the enzyme activity after addition of these compounds in percentage? (5 marks) F) The compounds in sections A) and B) were then removed. Name the technique used to remove these compounds. (5 marks) G) Anfinsen then used air in the oxidation of the disulphide bonds. What was the enzyme activity after this process in percentage? (10 marks) H) Indicate TRUE or FALSE. What significant conclusion or conclusions did Anfinsen draw from this FIRST EXPERIMENT? (10 marks) i) That the sequence of a protein is unique to that protein ii) That the Primary structure of a protein was the sequence of residues iii) That Primary structure dictates Tertiary structure iv) That Native proteins are in their most thermodynamically stable structure v) That cysteine residues form disulphide bonds

3 I) EXPERIMENT TWO. Anfinsen now conducted a second experiment. Fill in the blanks from the list of choices given below. (50 marks). Anfinsen added the two compounds identified in A) and B) above and once again they affected the RNAse A. He then removed of these compounds by the process identified in section F) and this enabled to form. If assayed at this stage the enzyme would have activity. By performing the next process in the experiment Anfinsen allowed to be reformed. After this process, when assayed, the enzyme now had activity. Anfinsen concluded that the disulphide bonding was important in the structure of RNAse A because with four cysteine residues in the sequence, there were possible arrangements that could form randomly, and only one would be correct. The rest of the structures he termed. Anfinsen then added a of a compound specifically to make small alterations to the disulphide bond arrangements. Over a period of around 10 hours the structure rearranged. When the enzyme activity was taken after this it was found to be active. Anfinsen drew more conclusions from this experiment. He said that the most stable structure of a protein is its Native structure. thermodynamically; both; the disulphide bonds; trace amount; one; zero percent; 100 percent; the Native structure; ionically; 105; 24; 10 percent; scrambled RNAse A; about one percent; radically; 10; the non-covalent bonds; 45 percent; glycolytically; all the covalent bonds

4 (100 marks = 33%) 4. The citric acid cycle is as its name suggests a cycle. Answer the following sections: A) The citric acid cycle is also known as: (5 marks) The Krebs cycle or B) Fill in the blanks. (20 marks) Prior to the citric acid cycle the enzyme complex Pyruvate dehydrogenase performs the reaction: Pyruvate CO 2 + C) Fill in the blanks. (10 marks) The enzyme Citrate synthase performs a condensation reaction: + oxaloacetate + H 2 O + Citrate D) Fill in the blanks. (15 marks) In the citric acid cycle, the enzyme converts: Citrate into via the intermediate

5 E) Fill in the blanks from the list of choices given below. (50 marks). In the citric acid cycle the enzyme converts to succinyl-coa. This enzyme process is one of two steps in the cycle the other being the previous step in the cycle carried out by the enzyme. In each of these processes the coenzyme is converted to. The enzyme converts succinate to the molecule. This molecule is then hydrated by the addition of water to form malate by the enzyme. From this product by an process we then arrive back at oxaloacetate. It is this molecule that is involved in the initial processes of the citric acid cycle and proves that the process is indeed cyclic. NADH; alpha-ketoglutarate dehydrogenase; FADH 2 ; CO 2 ; ornithine transcarbamoylase; citrate synthase; oxidation; NAD + ; succinate dehydrogenase; oxidative decarboxylation; chymotrypsin; isocitrate dehydrogenase; fumarate; FAD; dehydration; alpha-ketoglutarate; citrate; argininosuccinate synthetase; fumarase; isocitrate