BIO th November 2002 KEY EXAM III
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1 1 BIO th November 2002 KEY EXAM III This exam may be taken apart for grading. Please PRINT your name on each page. If you do not have sufficient room for your answer in the space provided, please continue on the back of the page on which the question appears. Question Maximum Points Earned Points I II III IV V VI VII VIII IX X XI TOTAL 100 NOTE: FULL CREDIT WILL BE GIVEN FOR THOSE ANSWERS WHICH CLEARLY ADDRESS ALL RELEVANT ASPECTS OF THE QUESTION S IN THE CLEAREST AND MOST CONCISE MANNER.
2 Name 2 I. [11 points; The point count assignment corresponds to the number of correct answers expected.] Illustrate your familiarity with fatty acid synthesis and oxidation by matching the reactant or characteristic in the right hand column with the appropriate pathway in the left hand column. a) Fatty acid oxidation 1,2,3,7 1) Acyl-CoA b) Fatty acid synthesis _1,4,5,6,8,9,10 2) Occurs in the mitochondria 3) NAD + -dependent 4) Three unique thiols are involved 5) ACP involved 6) Requires cooperation between sites on two identical chains of a dimer. 7) FAD-dependent 8) NADPH-dependent 9) Committed step is regulated by hormone-mediated protein phosphorylation 10) Malonyl-CoA
3 Name 3 II. [10 points] Identify and state the metabolic/biochemical significance of the items listed below; your answers must be consistent with material covered in class or assigned in the text. Choose FIVE; only the first five answers will be graded. The space allotted does not indicate the amount of information that may be needed for a proper answer. a) serine hydroxymethyl transferase Please see the text. b) HMG-CoA c) Carbamoylphospate Synthetase-1 d) SAM e) ACP f) Methylmalonyl-CoA g) Vitamin B 12 h) Vitamin B 6 i) Superoxide dismutase j) ApoCII k) N-Acetyl glutamate l) Branched-chain aminotransferase m) Hormone-sensitive lipase n) Carnitine o) ATP synthase p) ApoE q) Ubiquinone r) Proteasome s) Ubiquitin t) Reversed electron flow PLEASE ANSWER ON THE BACK OF THIS PAGE.
4 Name 4 III. [5 points ] List (by appropriate letter) the steps of one cycle of fatty acid synthesis in their correct order, beginning with the earliest step given below. PLEASE DO NOT DRAW ARROWS BETWEEN THE ITEMS AS GIVEN BELOW. A) The double bond is reduced to form butyryl-acp B) Condensation of acetyl and malonyl groups produces acetoacetyl-acp C) Water is removed to yield a double bond D) The carbonyl group of acetoacetyl-acp is reduced E) The fatty acid synthase is charged with correct acyl groups E > B> D> C> A IV. [10 points]. After the birth of a lamb, the udder of a ewe uses almost 80% of the total glucose synthesized by the animal. The glucose is used for milk production, principally in the synthesis of lactose and and glycerol-3-phosphate used for the synthesis of milk triacylglycerols. During the winter when food quality is poor, milk production decreases and the ewes sometimes develop ketosis. 1. Why do the ewes develop ketosis? ( 5 pts ) With limited carbohydrate intake, $-oxidation of FA's produces more acetyl-coa than can be oxidized by the citric acid cycle,due to diminished saturation of the enzymes in the cycle. Ac-CoA units are then shunted disproportionately into ketone body production. 2. A standard treatment for sheep ketosis is the administration of propionate. How does this treatment work? ( 5 pts ) Propionic acid is readily converted into succinate, thus raising the level of cycle intermediates, and also contributing to gluconeogenesis.
5 Name 5 V. [ 8 points] When O 2 is added to an anaerobic suspension of cells consuming glucose at a high rate, the rate of glucose consumption declines dramatically, O 2 is consumed and accumulation of lactate ceases. This effect, first observed by Louis Pasteur in the 1860s, is characteristic of most cells capable of both aerobic and anaerobic glucose catabolism. Respiration-deficient yeast mutants (P - ; "petites") can be produced from wild-type parents by treatment with mutagenic agents. The mutants lack cytochrome oxidase, a deficit that markedly affects their metabolic behavior. These mutant strains do not exhibit a Pasteur effect. Why does the absence of cytochrome oxidase eliminate the Pasteur effect? [Hint: Start by stating concisely why normal cells (as in the first paragraph and wild type yeast) exhibit the oxygeninduced reduction in glucose consumption.] Aerobic catabolism of glucose yields more ATP/glucose molecule than anaerobic metabolism. When cells acquire sufficient ATP there are regulatory mechanisms (e.g. inhibition of PFK-1 by ATP) that lower the rate of glucose consumption. Oxygen consumption accelerates the rate at which this state is attained. In the absence of cytochrome oxidase (the terminal step in the ETS) oxygen coupled consumption and ATP synthesis are not possible.
6 Name 6 VI. [ 8 points] When DCCD is added to a suspension of tightly coupled, actively respiring mitochondria, the rates of oxygen consumption and ATP synthesis dramatically decrease. If 2,4-dinitrophenol [DNP] is added, O 2 consumption resumes but ATP synthesis remains inhibited. 1. What is the molecular basis of the effect of DCCD? (3 pts) DCCD binds to a crucial glutamyl side chain on Fo, blocking proton flow through ATP synthase. 2. Why does DCCD affect O 2 consumption? (2 pts) In intact mitochondria, oxygen consumption, ATP synthesis, and electron transport are very tightly coupled. Blocking proton flow (and thus ATP synthesis) blocks oxygen consumption. 3. Explain the described effect of DNP? (2 pts) DNP causes collapse of the proton gradient and restores electron transport and oxygen consumption. It uncouples the system. 4. Which of the following inhibitors does DCCD most resemble in its mode of action? Antimycin A, rotenone, cyanide, or oligomycin (1 pt) Oliogomycin
7 Name 7 VII. [12 points] Answer A OR B, but not both; only your first answer will be graded. A. Sonication of mitochondria produces submitochondrial particles derived from the inner mitochondrial membrane. The membranous vesicles seal inside out, so that the inter-membrane space of the mitchondrion becomes the lumen of the submitochondrial particle. 1. Diagram the process of electron transfer and oxidative phosphorylation in these particles. (6 pts) It was expected that you outline the sequence of events similar to Figure in a closed sphere with the F1 portion of Complex V on the outside. 2. Assuming all the substrates for oxidative phosphorylation are present in excess, does ATP synthesis increase or decrease with an increase in the ph of the fluid in which the submitochondrial particles are suspended? Explain. (6 pts) In this orientation, an increase in ph in the external medium creates a gradient of protons that is comparable to that in an intact mitochondrion. Thus ATP synthesis should increase. 1 B on next page
8 Name 8 B. Noji et al (1997) performed an elegant experiment that allowed the direct observation of the rotation of the ((gamma) subunit of F 1 -ATPase relative to " 3 $ 3. [A b/w figure illustrating the experimental setup was included in Handout 13.] In a subsequent report they performed a similar experiment in which " 3 $ 3 was linked to a bead via a His tag instead of directly to the cover slip, and a lower ATP concentration was used to drive the rotation, otherwise the experimental setup was essentially the same. The following paragraph is taken from this second paper. A single molecule of F 1 -ATPase, a portion of ATP synthase, is by itself a rotary motor in which a central ( subunit rotates against a surrounding cylinder made up of " 3 $ 3 subunits. Driven by three catalytic $'s, each fueled by ATP hydrolysis, the ( subunit makes discrete 120 o steps. The work done in each step is constant over a broad range of imposed load and is close to the free energy of hydrolysis of one ATP molecule. What does the ATPase activity of F 1, together with the phenomenon of reversed electron flow in intact mitochondria, suggest about the relevance of studies such as the one described above to the mechanism of ATP synthase? [Hint: As PART of your answer, it might be useful to describe the binding change mechanism that is proposed for ATP synthase.] As described on pages of your text, the mechanism postulates a rotation of the " 3 $ 3 with respect to the central γ subunit. The subunits exhibit three different conformational states for binding of ADP and Pi and subsequent release of ATP. The energy for the process is from the flow of protons through the central core of F 0. F 1, in combination with F 0, functions as an ATP synthase. F 1 alone functions as an ATPase. Reversed electron flow by intact mitochondria is driven by the hydrolysis of ATP. The implication is that intact ATP synthase can operate in both the hydrolytic and synthetic directions, depending on the experimental conditions. The experiment described above, in which the hydrolysis of three successive ATP s drive the complete,360 o, rotation of F 1 is consistent with the binding change mechanism operating in reverse.
9 Name 9 VIII. [11 points] Some individuals have normal levels of phenylalanine hydroxylase, but an exceptionally low value for the K M of dihydrobiopterin reductase for dihydrobiopterin. Would you expect these individuals to excrete normal, elevated, or low concentrations of phenyllactate and phenylpyruvic acid? Explain in detail. Among other items it is essential that you: 1) state the function of phenylalanine hydroxylase. [1 pt] It catalyzes the first step in Phe catabolism and the last step in Tyr synthesis. 2) state the relevance of dihydrobiopterin to this question. [4 pts] F hydroxylase requires tetrahydrobiopterin; the reaction converts it to dihydrobiopterin. In order for the F hydroxylase to continue to function the dihydrobiopterin must be reduced back to the tetrahydro form. 3) state the significance of a low K M for dihydrobiopterin. [4 pts] The increased K M means that the DHB reductase has a higher apparent affinity for dihydrobiopterin and the degree of saturation of the enzyme is greater at lower [DHB]. This means that conversion of DHB to THB proceeds to a greater extent than in nomal individuals. 4) use this information to predict whether these individuals will excrete abnormal levels of phenylalanine metabolites. Your rationale must be clear. [2 pts] They will excrete diminished levels of phenyllactate and phenylpyruvate because the normal pathway is more effective.
10 Name 10 IX. [11 points] Cholesterol and other lipids are transported in the human body by lipoproteins. Indicate whether each of the following statements about these molecules is true (T) or false (F). T a) The density of a lipoprotein is inversely proportional to the lipid content. T b) Lipoptoteins "solubilize" hydrophobic lipids by surrounding them with a monolayer of amphipathic lipids. F c) Endogenous (internally synthesized) triacylglycerols [TG's] follow the same route in the body as exogenous TG's (obtained from the diet). T d) Both cholesterol and cholesterol esters are transported in lipoproteins. F_ e) Chylomicrons are the largest but least dense lipoproteins, containing a low proportion of TG's. T f) Chylomicrons carry fatty acids obtained in the diet to tissues for use or storage. T g) VLDL perform a role similar to that of chylomicrons, but with endogenous lipids rather than dietary lipids. F h) As TG's are removed from VLDL's by lipoprotein lipase, the VLDL's become chylomicrons. T i) VLDL's, IDL's, LDL's and HDL's are progressively denser versions similar, but non-identical molecular species. T j) LDL's are very high in cholesterol and cholesterol esters. T k) The liver is the only route through which cholesterol leaves the body (as bile salts lost in the digestive process.
11 Name 11 X. [ 6 points] The following summary is taken from Plump et al. (1992) Cell Explain why the absence of ApoE is associated with rapid onset of atherosclerosis. The paragraph is separate but the question has all you need to know for answering the question. ApoE facilitates the uptake of LDL and other lipoprotein particles by the liver, where cholesterol is converted to bile salts and is subsequently removed from the body. Without ApoE, therefore, the circulating levels of cholesterol are elevated.
12 Name 12 XI. [8 points] Match the product on the left with the appropriate precursor or characteristic from the list on the right. a) Serine f 1. Carbon chain from a three carbon intermediate from glycolysis and sulfur from methionine b) NO e 2. A derivative of tyrosine c) Urea b,c 3. Arginine is the precursor d) Proline g 4. Derived from a citric acid cycle intermediate by transamination e) Thyroxine d 5. A derivative of glutamic acid f) Cysteine a 6. Derived from a glycolytic intermediate g) Aspartic acid h 7. Derived from tryptophan h) Serotonin.
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