BCH 4054 PRE-TEST 1 GROUP September 18, 1995 NAME _____________________ This test is take-home and open book, and it is intended that all members of the group contribute to completing it. Only one copy is to be submitted by the group, and all members who participated should sign their names below. Test is due by 1:30 pm on Monday, September 23. __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ (16) 1. Under some conditions the liver oxidizes fatty acids to acetoacetate, which is secreted into the blood and then oxidized in peripheral tissues (such as heart muscle) to produce energy. (a) Give the overall pathway, indicating by name or structure all intermediates, by which palmitic acid (C16:0) is oxidized completely to CO2 in this fashion, distinquishing which reactions occur in liver and which in heart muscle. (b) Identify the steps at which ATP, GTP, NADH, and CoQH2 are utilized or produced. (c) Assuming reoxidation of NADH and CoQH2 by the electron transport chain, calculate the net yield of ATP in liver and in heart muscle from this overall reaction when 2.5 ATP are made for each NADH, 1.5 ATP from CoQH2, and 1 ATP from each GTP. (continue answer on back of page if necessary). (16) 2. Oxidation of glutamine in the liver would show the overall stoichiometry: glutamine -----> 4 CO2 + urea (a) Give the overall pathway by which this reaction would occur in liver, indicating by name or structure all intermediates. (b) Identify which steps occur inside the mitochondria and which occur in the cytoplasm. (c) Identify the steps at which ATP, GTP, NADH, and CoQH2 are utilized or produced. (d) Assuming reoxidation of NADH and CoQH2 by the electron transport chain, calculate the net yield of ATP in liver from this overall reaction when 2.5 ATP are made for each NADH, 1.5 ATP from CoQH2, and 1 ATP from each GTP. (continue answer on back of page if necessary). Use the standard reduction potentials found in Table 13-7, page 388, and delta Go' of hydrolysis from Table 13-6, page 377. Use the following constants in your calculations: R = 8.3 x 10-3 kJ mol-1 K-1; ln x = 2.3 log x; T = 310 K; F = 96.5 kJ volt-1 equiv.-1 3. (a) Calculate delta Go' and K'eq for the following reaction: (5) NADH + fumarate -----> NAD+ + succinate (4) (b) This reaction can be catalyzed by submitochondrial particles, created by sonication of mitochondria in which the inner mitochondrial membrane recloses inside out to form closed spherical vesicles. Diagram such a particle, showing the location and orientation of each protein complex involved in the catalysis and how they participate in the reaction. Identify the intermediate electron carriers (A,B,C,D,E) involved in the reaction. NADH --> A --> B --> C --> D --> E --> fumarate (4) (c) Such vesicles as described above should pump protons from the solution to the interior of the vesicle. Assuming the proton stoichiometry proposed for the complexes involved, calculate the proton motive force (delta p) that would be created if the NADH/NAD+ and fumarate/succinate ratios were each maintained at 1:1. (4) (d) If the bulk solution were maintained at pH 7.0 in this experiment, and the electrical potential (delta psi) across the vesicle was determined to be 0.10 volt, what should the internal pH of the vesicle be? 4. Isolated mitochondria can carry on a number of other partial reactions related to oxidative phosphorylation. For example, the oxidation of added cytochrome c by oxygen is coupled to the formation of ATP, yielding the following reaction: 2 cyt c (red) + 1/2 O2 + ADP + Pi ----> 2 cyt c (ox) + H2O + ATP (3) (a) This reaction can be carried out by mitochondria, but would not work with the inside out submitochondrial particles described in question 3. Explain why not. (4) (b) What protein complexes and intermediate carriers must be present in the mitochondrial membrane for this overall reaction to be catalyzed? (Remember, the reactants are being supplied to the bulk solution, which is external to the mitochondrial membrane). (4) (c) Calculate delta Go' for the coupled reaction above in 4(a) (i.e., with reactants and products in their standard states). (6) (d) How would each of the following inhibitors affect the reaction in 4(a)? cyanide antimycin dinitrophenol rotenone oligomycin valinomycin + K+ 5. ATP synthesis in chloroplasts is coupled to proton transport as it is in mitochondria, but there are some differences from the mitochondrial system. (3) (a) The ATP synthase is on the matrix side of the inner mitochondrial membrane. Describe its location and orientation in the chloroplast. (3) (b) Electron transport pumps protons out of the mitochondrial matrix into the space between the inner and outer membranes. Between what spaces and across what membrane is the gradient formed in chloroplasts? (3) (c) A cytochrome complex in mitochondria catalyzes transfer of electrons from coenzyme Q, a quinone, to cytochrome c, a protein, and consists of cytochromes b and c1 and an iron- sulfur protein. The cytochrome complex in photosynthesis catalyzes transfer of electrons from what quinone to what protein, and consists of what cytochromes? (3) (d) The proton-motive force generated in mitochondria involves both a pH and an electrical potential gradient across the membrane. What is different in the proton-motive force in chlorplasts? (10) 6. The light reaction of photosynthesis involves two separate pigment systems. For each of the following statements, indicate in the blank whether the statement refers to pigment system I (P.S.I), pigment system II (P.S.II), both, or neither. ________ Contains a pigment with an absorption maximum of 680 nm. ________ Absorption of light produces a strong reductant and a weak oxidant. _______ Contains antenna chlorophyl. ________ Contains a manganese cluster. ________ Is inhibited by DCMU. ________ Is involved in cyclic photophosphorylation. ________ Is concentrated along the stromal surface of the thylakoid membrane. ________ Contains bacteriorhodopsin. ________ oxidizes reduced plastocyanin. ________ contains pheophytin. (12) 7. Three vitamins are involved as precursors of tightly bound prosthetic groups in the catabolism of fatty acids and amino acids: vitamin B6, vitamin B12, and biotin. For each vitamin, give the name of the prosthetic group, indicate whether it is covalently bound to the enzyme, and identify a reaction it is involved in (including name of enzyme, reactants, and products).