EXAM 3a BIOC 460 Wednesday April 10, 2002 Please include your name and ID# on each page. Limit your answers to the space provided! 1
1. (5 pts.) Define the term energy charge: Energy charge refers to the ratio of ATP to ADP and AMP. The term is defined as: Energy Charge = [ATP] + 0.5 [ADP] [ATP] + [ADP] + [AMP] 2. (5 pts.) How many moles of ATP are generated from the catabolism of two moles of glucose under anaerobic conditions? Under anaerobic conditions, catabolism of two moles of glucose produces 4 moles of ATP. 3. (5 pts.) Explain in biochemical terms why an individual with a deficiency in the enzyme lactate dehydrogenase has difficulty running up a flight of stairs. Lactate dehydrogenase converts pyruvate to lactate and is required to replenish the necessary NAD+ needed for the energy production phase of glycolysis [at the step of glyceraldehyde 3-P dehydrogenase]. Therefore, strenuous anaerobic exercise cannot be maintained in these individuals because glycolysis is blocked and the muscles are depleted of the necessary ATP needed for contraction. 4. (5 pts.) Adults usually express only low levels of the enzyme lactase in their intestines and are considered lactose intolerant. What explains the gastrointestinal problems associated with the ingestion of milk products in these individuals? Without the enzyme lactase, ingestion of milk products leads to lactose build-up in the intestine which is metabolized by the bacterium lactobacillus. The products of this fermentation reaction are gas and lactic acid which cause stomach problems. 5. (5 pts) Explain why it makes sense that the enzyme pyruvate kinase is activated by fructose 1,6-bisphosphate when blood glucose levels are high. Pyruvate kinase converts phosphoenolpyruvate [and ADP] to pyruvate [and ATP] in the last step of the glycolytic pathway. When blood glucose levels are high, cells metabolize glucose to form glycolytic intermediates such as fructose 1,6- bisphosphate, which in turn, activates pyruvate kinase to stimulate metabolic flux in the direction of pyruvate formation. 2
6. (5pts.) Name the glycolytic enzyme that opposes the reaction in gluconeogenesis catalyzed by fructose 1,6-bisphosphatase? Does citrate activate (+) or inhibit (-) this glycolytic enzyme? Phosphofructokinase is the glycolytic enzyme that opposes the gluconeogenic enzyme fructose 1,6-bisphosphatase [these enzymes interconvert fructose 6-P and fructose 1,6-BP]. Citrate inhibits phosphofructokinase. 7. (5 pts.) The liver mitochondrial enzyme pyruvate carboxylase catalyzes the reaction: Pyruvate + CO 2 + ATP + H 2 O Oxaloacetate + ADP + P i + H + What is most of the oxaloacetate converted into under the following conditions; a) when blood glucose levels are low and the energy charge is high? b) when mitochondrial acetyl CoA levels are high and energy charge is low? When blood glucose levels are low the oxaloacetate is converted to PEP and used in the gluconeogenic pathway to produce glucose for release into the blood. When mitochondrial acetyl CoA levels are high, the oxaloacetate is converted to citrate in the citric acid cycle to meet the energy needs of the cell. 8. (5 pts.) Write the net reaction of the nine steps of the Citric Acid Cycle. Acetyl-CoA + 3 NAD + + FAD + GDP + P i + 2 H 2 0 ---> 2 CO 2 + 3 NADH + FADH 2 + GTP + CoA [+ 2 H + ] 9. (5 pts.) Why do individuals with the neurological disorder beriberi contain a large amount of pyruvate in the blood after eating a high carbohydrate meal? Beriberi is nutritional deficiency in thiamine pyrophosphate (vitamin B1) which is a required cofactor in the enzyme pyruvate dehydrogenase. Individuals with beriberi cannot readily metabolize pyruvate to acetyl CoA because pyruvate dehydrogenase is inactive without thiamine pyrophosphate. This leads to a build-up of pyruvate in the blood under conditions when glycolysis is stimulated. 10. Early biochemists used radioactively labeled 14 C-acetyl CoA in an in vitro system to show that the Citric Acid Cycle was indeed a cycle. a. (5 pts) What fraction of CO 2 released during one turn of the cycle was found to contain radioactive 14 C? Explain. None of the CO 2 released in the first turn of the cycle was radioactive because all of it was incorporated into cycle intermediates [a minimum of two turns is needed]. 3
b. (5 pts.) Explain why no radioactive carbon was found in oxaloacetate when inorganic phosphate (P i ) was removed from the system prior to adding 14 C-Acetyl CoA? Inorganic phosphate is required by the CAC reaction step that couples GTP synthesis to thioester bond cleavage [succinyl CoA synthetase]. Without Pi, this enzyme reaction is inhibited and radioactive carbon would only be found in cycle intermediates that precede this reaction step [radioactive carbon was not found in succinate, fumarate, malate or oxaloacetate]. 11. (5 pts.) What explains the observation that oxidative phosphorylation produces 2.5 moles of ATP per mole of NADH derived from citric acid cycle reactions, whereas in the kidney and brain, only 1.5 moles of ATP are synthesized per mole of NADH produced by glycolysis? Oxidation of NADH generated inside the mitochondrial results in the pumping of 10 H+ out of the mitochondrial matrix. Since 4 H + are required for each ATP synthesized by ATP synthase, this produces 2.5 ATP. However, all tissues except the liver and heart muscle, NADH derived from the glyceraldehyde 3-P dehydrogenase reaction in glycolysis must be shuttled into the mitochondria by the glycerol 3-P shuttle. This shuttle results in the reduction of 1 FAD for every NADH oxidized. Since oxidation of FADH 2 by the electron transport chain occurs at a step downstream of NADH oxidation [FADH2 is oxidized by complex 2], this results in the pumping of only 6 H+ out of the mitochondrial matrix and hence only 1.5 ATPs. 12. (5 pts.) Why does it make sense that a dramatic increase in cytosolic levels of cytochrome c functions as an initiating signal for cell death (apoptosis) in eukaryotes? Cytochrome c is required for electron transport and is localized to the inner mitochondrial membrane, it is not supposed to be in the cytosol. The presence of high levels of cytochrome c in the cytosol signals that the mitochondria are not functioning properly and that the cell will, or needs to, die. 13. (10 pts.) An ATP synthase complex has been characterized that contains a c ring with 9 identical c subunits. Experiments have shown that 1 ATP is synthesized for every 3 H + that move through the complex into the matrix. Explain this 1:3 ratio in terms of the structure and function of the gamma and beta subunits of ATP synthase. When the H + binds to a single c subunit, it causes a clockwise rotation of the c ring 40 degrees (1/9 of a circle). The three-sided gamma subunit rotates along with the c ring and interacts directly with the three beta subunits. For every 120 degree rotation of the c ring, the gamma subunit induces the O, T or L conformation in each of the beta subunits. Since an ATP is synthesized every time the gamma subunit completes a turn of 120 degrees [T to O conformational change in a beta subunit], it requires 3 H + moving through the complex in order to synthesize 1 ATP [3 x 40 degree rotation]. 4
14. (5pts.) What happens to the rate of oxygen consumption when dinitrophenol (DNP) is added to a suspension of mitochondria that have been pretreated with cyanide? Explain. Cyanide blocks the electron transport chain, resulting in loss of the proton gradient and shut down of ATP synthesis. Addition of DNP to this system will have no effect on oxygen consumption because cyanide is still blocking the electron transport chain. 15. (10 pts.) The diagram below illustrates the photosynthetic process in plants. Cross out each component in A through E that does not accurately describe photosynthesis. There should only be five crosses when you are done. 16. (5 pts.) Carbon dioxide is a C 1 substrate in the Calvin Cycle reaction catalyzed by the enzyme Rubisco. What is the one C 5 substrate and two C 3 products of this reaction? The mass of Rubisco on planet earth is higher than that of any other enzyme, why? The C 5 substrate is ribulose 1,5-bisphosphate and the C 3 products are two moles of 3- phosphoglcerate. Rubisco is a very large multisubunit enzyme that is found in all plants, most notably, marine photosynthetic organisms. 17. (5 pts.) Why do individuals with a deficiency in the enzyme glucose 6-P dehydrogenase become anemic if they are treated for malria with pamaquine? A deficiency in glucose 6-P dehydrogenase results in low amounts of NADPH in blood cells because this enzyme catalyzes the first step in the pentose phosphate pathway. Low amounts of NADPH inhibits the glutathione reductase reaction and thus lowers the level of reduced glutathione needed to inactivate pamaquine which is toxic to red blood cells. As a result, the red blood cells lyse and the individual becomes anemic. 5