(3) After the first reaction releasing CO2, the following oxidation in the mitochondria generates the bulk of the ATP. Please
concisely describe the process how the oxidation energy is captured and converted into ATP. (8%)

1. A scientist uses the yeast expression system to over-express one recombinant protein, and which is also an enzyme. Based on the enzymatic activity of the recombinant protein, the scientist is able to purify the protein and makes the purification table. Purification procedure Total protein (µg) Enzymatic activity (units) 1. Crude extract 60,000 1,000,000 2. Sodium sulfate precipitation 30,000 700,000 3. Affinity chromatography 5,000 520,000 4. Ion-exchange chromatography 3,800 376,000 (1) From the information given in the table, please calculate the specific activity of the enzyme after the final procedure. (2%)

(2) Which purification procedure used for this enzyme is the most effective? What is the principle of the procedure? (4%)

(3) To verify the identity of the purified recombinant protein, which assay would you suggest to use? (2 %)

(4) The protein is composed of 420 amino acids.What is the approximate molecular weight of the protein? (2%)

(5) When the protein is resolved on the sodium dodecył sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie brilliant blue R250 (CBR) staining, only a protein band around 55 kDa is observed on the gel. Do you think the result match the predicted molecular weight of the protein from (4)? If not, please explain the possible reason for that. In addition, which experiment would confirm that? (4%)

(6) When the protein is resolved on the Native-PAGE followed by CBR staining, three protein bands are observed on the gel. Please explain these three protein bands. (2%)

(7) The enzyme has three substrates, including substrate A, B & C. The K, of substrate A, B and C to the enzyme is 8 nM, 50 nM and 4 mM, respectively. Among these three substrates, which one has the best reaction with the enzyme? Why? (4 %)

2. In cellular energy production process, the transformation of glucose to lactate in myocytes (muscle cells) releases only about 7% of the free energy produced when glucose is completely oxidized to CO2 and H2O. (1) Does this mean that anaerobic glycolysis in muscle is a wasteful use of glucose? Please explain. (4%)

(2) Under aerobic conditions, all six carbons of glucose are oxidized to CO2. What is the first reaction that releases CO2? (Please include the name of the enzyme.) (4%)

(4) Compound X is an inhibitor of mitochondrial ATP synthesis. It was observed that when compound X was added to cells, the NAD*/NADH ratio decreased. Would you expect X to be an uncoupling agent or an inhibitor of the respiratory chain? Please explain. (4%)

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