Metabolic Biochemistry / BIBC 102 Midterm Exam / Spring 2011 I. (25 points) Fill in all of the enzyme catalyzed reactions which convert glycogen to lactate. Draw the correct structure for each intermediate molecule, showing the correct position of the hydrogen atoms at each carbon atom. Include the names or abbreviations of any other reactants and products (eg, ATP, ADP, Pi, NAD+, NADH, etc), and any required prosthetic groups (eg, biotin, TPP, etc.). (Note: you do not need to give the correct name for each intermediate molecule or the correct name of the enzyme to receive full credit. Partial credit may, however, be given for the correct name of an intermediate structure if the structure itself is incorrect.) Glycogen (draw the 2 terminal sugar subunits of the polymer only) lactate (draw structure)
II. (25 points) A. Show how pyruvate is converted to oxaloacetate by pyruvate dehydrogenase plus the enzymes of the TCA cycle. Draw the correct structure for each intermediate molecule, showing the correct position of the hydrogen atoms at each carbon atom. Include the names or abbreviations of any other reactants and products (eg, ATP, ADP, Pi, NAD+, NADH, etc), and any required prosthetic groups (eg, biotin, TPP, lipoate, etc.). B. Show the path of 14 C label in each molecule starting with 14 C label in the carbonyl carbon of pyruvate (CH 3 -- 14 C(O)--CO 2 -). 2
3 III. (25 points) (A) Draw a diagram of the overview of proton and electron circuits in mitochondria starting with NADH in the matrix and ADP and P i in the cytosol and ending with the reduction of oxygen to water and the formation of ATP (the diagram in the lecture handout will receive full credit). Label each complex and protein, identify matrix, intermembrane space, and inner membrane, and indicate the flow of protons and electrons. (B) Indicate on this diagram the sites of action for rotenone, antimycin, oligomycin, atractyloside, and dinitrophenol (DNP).
4 IV. (10 points). When oxygen is removed from a suspension of yeast cells using glucose for energy, the rate of glucose consumption increases dramatically and ethanol begins to accumulate in the media. This effect, first observed by Pasteur, is characteristic of most cells capable of both aerobic and anaerobic utilization of glucose. A. Why did ethanol begin to accumulate when oxygen was removed? B. What regulatory mechanism(s) explain how the removal of oxygen increases the rate of glucose consumption? Explain in terms of the action of allosteric effector(s) on specific enzyme (s). V. (10 points) When a suspension of intact mitrochondria are incubated in a buffer containing oxygen, phosphate, and an abundant source of matrix NADH, no oxygen is consumed. When 50 µmoles of ADP are added to this mitochondrial suspension, there is a period of rapid oxygen consumption that is followed by no oxygen consumption. A. Explain the molecular basis for the effect of ADP on oxygen consumption in this system. B. Why did the period of oxygen consumption end? C. How many moles of oxygen (O 2 ) were consumed?
VI. (15 points) (A) In the space below, please sketch the lineweaver burke plot for an enzyme in the presence and absence of a competitive inhibitor when the inhibitor concentration is [I] = Ki. Label each axis and show the values of each intercept in terms of Km, Vmax, and Ki. With reference to your plot, briefly explain how competitive inhibitors inhibit enzyme activity. 5 (B) In the space below, please sketch the Vo versus S plot for an enzyme which shows positive cooperativity in substrate binding, in the presence and absence of an allosteric inhibitor; label each line and axis. With reference to this plot, explain how the allosteric inhibitor decreases the activity of the enzyme at the physiological concentration of substrate found in a cell.
6 VI. (90 points) Multiple Choice. Choose only one answer for each question. 1. Which one of the following statements about the ribbon structure of the enzyme depicted below is blatantly false? (A) There are 8 strands in a parallel β sheet structure. (B) There are 8 α helices. (C) The arrow points to the amino terminus of this protein. (D) Amino acids E, Q, and N are far more likely to be found in an α helix of this protein than in a β strand. (E) If the R groups of the amino acid residues in positions 1,3,5, and 7 in a β strand of this protein project to the interior of the β sheet plane (actually the interior of the β barrel), the R groups of the amino acid residues is positions 2,4, and 6 will project to the exterior of the β sheet plane (the outside of the β barrel). 2. You will find below a pentapeptide with correctly drawn structures of five amino acids. Which one of the following five groups of amino acids is found in this pentapeptide? (A) Isoleucine, threonine, tyrosine (D) Serine, aspartate, glycine (B) Glutamate, tyrosine, glycine (E) Asparagine, phenylalanine, serine (C) Leucine, glycine, aspartate 3. Which one of the following statements is blatantly false? (A) Vmax, the maximal velocity, is the rate of product formation that is attained when all of the enzyme molecules have substrate bound to them. (B) An enzyme obeying Michaelis Menten kinetics will achieve 67% of Vmax when [S] = 2 Km. (C) If an enzyme increases the rate of A to B by 10 8, it will also increase the rate of B to A by 10 8. (D) Competitive inhibitors bind reversibly to the active site of an enzyme and thereby prevent binding of the substrate. (E) The velocity of an enzyme catalyzed reaction, Vo, is proportional to the enzyme concentration, [E], when [S] << Km but not when [S] >> Km. 4. Which one of the following statements is blatantly false? (A) For an allosteric enzyme, the substrate concentration at which half maximal velocity is attained, called K 0.5, will be higher if an allosteric activator is present. (B) When an allosteric inhibitor binds to the allosteric site on an enzyme, it shifts the R/T equilibrium to the T state. (C) The phosphorylation of a specific serine residue in each subunit of glycogen phosphorylase activates the enzyme by shifting the R/T equilibrium to the R state. (D) Irreversible enzyme inhibitors typically inactivate enzymes by forming covalent bonds with an active site amino acid. (E) Positive cooperativity in substrate binding requires that enzymes have at least two active sites.
7 5. The synthesis of the activated form of acetate (acetyl-coa) is carried out in an ATP-dependent process catalyzed by acetyl CoA synthase: Acetate + CoA + ATP acetyl CoA + AMP + PPi The pyrophosphate product (PPi) of this reaction is rapidly hydrolyzed to 2 Pi in a reaction carried out by pyrophosphatase. What is the G for this reaction when the final product is 2Pi (in kj/mol)? You are given: G for hydrolysis of acetyl CoA to acetate + CoA is -32.2 kj/mol; G for hydrolysis of ATP to AMP + PPi is -30.5 kj/mol; and G for hydrolysis of PPi to 2 Pi is -33.4 kj/mol. (A) +96.1 (B)+1.7 (C) -1.7 (D) -96.1 (E) -31.7 6. Coupling ATP cleavage to an unfavorable reaction. Consider a hypothetical biosynthetic reaction A B whose equilibrium constant K = [B] / [A] = 10-3. In vivo, this reaction is coupled to the hydrolysis of ATP: A + ATP B + ADP + Pi, whose equilibrium constant is 10 3. By what factor does coupling the formation of B to the hydrolysis of ATP increase the ratio of [B] to [A] when the phosphorylation potential in the cell ( [ATP] / [ADP][Pi]) is 500 and the phosphate concentration is 2 mm? (A) 5 x 10 8 (B) 10 9 (C) 2 x 10 9 (D) 5 x 10 9 (E) 10 10 7. Which one of the following statements is blatantly false? (A) At steady state in a typical cell, the reactions catalyzed by pyruvate kinase and phosphoglycerate kinase are essentially at equilibrium. (B) Adenylate kinase catalyzes the reaction: AMP + ATP 2 ADP (C) An increase in the phosphorylation potential (i.e., the [ATP]/[ADP][Pi] ratio) will increase the rate of a biosynthetic pathway. (D) Under steady state conditions in a typical cell, the ability of ATP hydrolysis to do useful work (e.g., drive biosynthetic reactions) is given by the actual free energy change, G, a value that is substantially more negative than the standard free energy of ATP hydrolysis, G = -30.5 kj/mol. (E) For the phosphofructokinase reaction in a typical cell, the ratio of the products (fructose-1, 6- bisphosphate and ADP; numerator) to the reactants (fructose-6-phosphate and ATP; denominator) is smaller than if the reaction were at equilibrium. 8. Which one of the following statements is blatantly false? (A) The net ATP equivalents produced per glucose subunit of glycogen in an anaerobic muscle cell is 3. (B) The net ATP equivalents produced from glucose in an anaerobic muscle cell treated with arsenate is 0. (C) If glucose is labeled at carbon 4, pyruvate will be labeled at the carboxyl carbon. (D) If glucose is labeled at carbon 6, ethanol will be labeled in the methyl carbon. (E) In a yeast cell that lacks triose phosphate isomerase, none of the pyruvate formed from glucose-5-14 C will be labeled. 9. Which one of the following statements is blatantly false? (A) Glycogenolysis and Glycolysis occur in the cytosol, not in mitochondria (B) Pyruvate dehydrogenase catalyzes the critical committed step in glucose catabolism, since pyruvate dehydrogenase is irreversible under physiological conditions, and since glucose can be made from pyruvate but not from acetyl CoA. (C) Pyruvate dehydrogenase and the citric acid cycle enzymes are found in mitochondria, not in the cytosol. (D) Glycogen is a glucose polymer found in the liver and muscle cells whose highly branched structure creates numerous terminal glucose residues with free C4 hydroxyl groups, each of which is a substrate for glycogen phosphorylase. (E) Lactate dehydrogenase and pyruvate carboxylase are found in the cytosol, not in mitochondria.
10. Which one of the following statements is blatantly false? (A) Glycogen phosphorylase is activated by phosphorylation. (B) Pyruvate dehydrogenase is activated by phosphorylation. (C) Phosphofructokinase is allosterically activated by AMP. (D) Hexokinase is allosterically inhibited by glucose-6-phosphate. (E) Pyruvate carboxylase is allosterically activated by acetyl CoA. 8 11. You are studying catabolism and electron transport in a minced tissue preparation of pigeon breast muscle which contains all of the enzymes of glycolysis and intact mitochondria. You already know that this tissue is capable of perfectly normal aerobic and anaerobic glycolysis. Furthermore the TCA cycle and electron transport can all function normally. Now in the presence of O 2 and glucose, you add an inhibitor of electron transport to the aerobically respiring tissue. Which one of the following possible results are you likely to see? 1. O 2 consumption falls dramatically. 2. All ATP synthesis from glucose ceases. 3. The rate of glucose consumption increases 4. The NADH / NAD ratio in the cytosol decreases. (A) 1 and 3 (B) 1, 2, and 3 (C) 2 and 4 (D) 4 (E) All of the above 12. The following graph represents the hypothetical results of an experiment in which the amount of O 2 consumed is measured over a time interval during which intact mitochondria are treated sequentially with five substances. The identity of these 5 substances can be inferred from their effect on the amount of O 2 consumed. Assume that P i and an abundant source of matrix NADH are present. (A) 1 = Arsenate 2 = Oligomycin 3 = DNP 4 = Antimycin A 5 = Succinate (B) 1 = ADP 2 = Oligomycin 3 = Arsenate 4 = Cyanide 5 = DNP (C) 1 = ADP 2 = Oligomycin 3 = DNP 4 = Rotenone 5 = Glycerol P (D) 1 = DNP 2 = Rotenone 3 = Succinate 4 = Cyanide 5 = Arsenate (E) 1 = ADP 2 = Atractyloside 3 = DNP 4 = Oligomycin 5 = Arsenate 13. How many ATP equivalents can be produced in the complete aerobic catabolism of one glycerol molecule by a muscle cell? Consider GTP to be equivalent to ATP. (Hint: The first step is the ATP-driven phosphorylation of glycerol to glycerol phosphate in the cytosol). (A) 14 (B) 13 (C) 12.5 (D) 11.5 (E) 15 14. Which one of the following statements is blatantly false? (A) Thiamine pyrophosphate is a prosthetic group of the E1 subunit of pyruvate dehydrogenase. (B) If tritium (T) is attached to the middle carbon of lactate, lactate dehydrogenase will catalyze the transfer of this tritium atom plus an electron pair to the nicotinimide ring of NAD+ to form NADT. (C) The prosthetic group for E3 subunit of pyruvate dehydrogenase is FAD. (D) In the pyruvate carboxylase reaction, biotin is first carboxylated by CO 2 at one active site in a reaction that is driven by the coupled hydrolysis of ATP, and then, at the other active site, the carboxyl group of carboxybiotin is transferred to pyruvate to form oxaloacetate. (E) The order in which products are produced in the pyruvate dehydrogenase mechanism is acetyl CoA, then CO2, then NADH.
9 15. Which one of the following statements is blatantly false? (A) If intact mitochondria are incubated with O 2, ADP, Pi, and a source of matrix NADH, the addition of antimycin will cause ubiquinone to become fully reduced and cytochrome c to become fully oxidized. (B) If intact mitochondria are incubated with O 2, Pi, and a source of matrix NADH, the addition of ADP would be expected to increase the NAD /NADH ratio. (C) The respiratory quotient (ratio of CO 2 generated/o 2 consumed) for the complete aerobic catabolism of pyruvate is 1. (D) The exergonic transfer of two electrons from NADH to UQ using complex I provides the energy to pump 4 H + out of the mitochondria. (E) If 14 C labeled NADH is added to a suspension of intact mitochondria in the presence of oxygen, ADP, and P i, NO 14 C radioactivity will appear in the mitochondrial matrix. 16. Which one of the following statements is blatantly false? (A) In the glycerol phosphate shuttle, the inner mitochondrial membrane glycerol phosphate dehydrogenase transfers electrons from its FADH 2 prosthetic group to ubiquinone. (B) In intact mitochondria, electron transport will cease when the energy it takes for electron transport to pump a proton against a concentration gradient equals the energy obtained from the exergonic transfer of the electron pair from NADH to oxygen. (C) The prosthetic groups of Complex IV are cytochromes a and a3, and copper atoms CuA and CuB. (D) The prosthetic groups of Complex I are FAD and FeS. (E) Complex IV accepts electrons one at a time from cytochrome c and then transfers 4 electrons to O2 in order to form 2 H2O without generating oxygen radicals or peroxides. 17. When a cell extract containing all of the enzymes of glycolysis and the TCA cycle is incubated aerobically with Glucose-6-14 C, what percentage of the original label will remain in oxaloacetate after three turns of the TCA cycle? (In this problem assume that any label which enters the cycle does so in the first citrate synthase step, and that the acetyl CoA entering in the second and later turns of the TCA cycle is unlabeled.) (A) 0% (B) 25% (C) 50% (D) 75% (E) 100% 18. You will find below several correctly drawn structures of important compounds in biochemistry. Which of the following are represented? (1) NADH (2) FMNH 2 (3) UQH 2 (4) lipoic acid (5) Fructose (A) 1,3,5 (B) 1,3,4 (C) 1,2,3 (D) 1 and 5 (E) 2 and 4