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PRACTICE SET 6 A. Questions on Lipid Metabolism and Glyoxylate Cycle 1. The hydroxy acid given below can be completely oxidized to acetyl-CoA by betaoxidation. Write the series of individual reactions that will accomplish this degradation. Then write the overall sum showing how much ATP, CoASH and NAD+ is required and how much NADH, acetyl-CoA, etc., are produced on a per molar basis. Use structural formulas for all substrates and products and standard abbreviations for cofactors (e.g., NAD+, ATP); balance all equations. H H 3C C CH2 H H C C (CH2)4 COO- OH 2. Calculate the net number of moles of energy-rich phosphate (ATP equivalents) that should be produced when one mole tricaproin is oxidized in liver mitochonridria completely to CO2 and H2O via beta oxidation and the carbons of glycerol are oxidized to CO2. Show all calculations. O CH2 O C O CHO C (CH2)4 CH3 (CH2)4 CH3 (CH2)4 CH3 O CH2 O 3. C When one mole of arachidonic acid is oxidized to CO2 and H2O by β-oxidation and the Krebs cycle, how many moles of NADH, FADH2, ATP, and CO2 are produced? Arachidonic acid is a C 20 fatty acid with double bonds at carbons 5, 8, 11, 14. 4. Compare and contrast the pathway by which fatty acids are degraded (betaoxidation) with the pathway by which fatty acids are synthesized from acetate. Cover such points as: (a) nature of the "activated" structures; (b) coenzymes involved; (c) stereochemistry of the intermediates; (d) the places in the living cells where the pathways occur; (e) any significant (major) structural differences in any intermediates involved; and (f) the amount of ATP required for the two pathways. Write your answers in table form. 5. Explain in detail why an animal cannot affect the net conversion of lipid to carbohydrate while a plant or microorganism can. Illustrate your answer with the key enzyme-catalyzed reactions involved. 6. Although lipid degradation can not result in the net accumulation of glucose, explain why glycogen and blood glucose become radioactive when 14C-acetate (14CH3COO-) is fed to experimental animals. 7. The condensation of butyryl-S-ACP with malonyl-S-ACP occurs in fatty acid synthesis when a C4-fatty acid is elongated to a C6-fatty acid. Please write the structure of the hypothetical seven-carbon-S-ACP b-keto acid intermediate that is formed prior to decarboxylation of the malonyl moiety. The structure of butyrylS-ACP is given. H 3C CH2 CH2 C S ACP O 8. One reaction of the glyoxylate cycle is formally analogous to the reaction catalyzed by citrate synthase of the Krebs cycle. Write the reaction of the glyxoylate cycle using structures and abbreviations for co-factors. B. Questions on Amino Acid Metabolism 9. Give one example of each of the following reactions -- using specific compounds known to undergo those reactions: a. b. Decarboxylation of an amino acid. Aminotransferase (transamination). 10. When an experimental animal is administered aspartic acid labeled with 15N, both nitrogen atoms in urea become labeled with 15N. Outline the reactions which account for this observation. Name the enzymes that feed N into the urea cycle, don’t name the urea cycle enzymes. 11. Name the cofactor and its corresponding vitamin commonly found in aminotransferase enzymes. Name the cofactor and its corresponding vitamin commonly found in one carbon transfers in amino acid metabolism and dTMP synthesis. 12. Write the reaction that would be catalyzed by an enzyme called carbamoylP:glycine carbamoyltransferase. 13. Trace the path of the nitrogen atom from glutamic acid in a test tube solution containing only the enzymes: glutamate dehydrogenase, carbamoylphosphate synthetase I, and carbamoyl-P: aspartate carbamoyltransferase. 14. What does ketogenic and glucogenic mean? 15. Write the equations for two enzyme-catalyzed reactions that involve the incorporation of inorganic nitrogen into an organic molecule. Name the enzymes. 16. Explain why plants have no need to produce urea and yet contain nearly all the enzymes of the urea cycle. 17. Why is the dietary requirement of turkeys for methionine reduced if their diet also includes cysteine? What if it also included homocysteine? C. Questions on Regulation 18. What are the effects in muscle of the following on the rates of glycogen synthesis and glycogen breakdown? a. inhibiting adenylate cyclase b. increasing the adrenaline concentration in the blood 19. Which of the following statements about hormones are correct? a. Insulin increases the rate of glycogen synthesis in the liver. b. Insulin is secreted in response to low levels of blood glucose. c. Glucagon and epinephrine have opposing effects on glycogen metabolism. d. The effects of insulin, glucagon, and adrenaline are mediated by cyclic AMP. 20. Place the following steps of the reaction cascade of glycogen metabolism in the proper temporal sequence. a. Phosphorylation by catalytic subunit of cAMP dependent protein kinase b. Formation of cAMP by adenylate cyclase c. Phosphorylation of glycogen phosphorylase b d. Hormone binding to target cell receptors 21. Caffeine inhibits cyclic nucleotide phosphohydrolases. What effect would this inhibition have on glycogen metabolism when epinephrine levels are dropping in the blood? 22. During a fight or flight situation, the release of adrenaline promotes glycogen breakdown in the liver, heart, and skeletal muscle. The end product of glycogen breakdown in the liver is glucose. In contrast, the end product in skeletal muscle is lactate. Why are different products of glycogen breakdown observed in the two tissues? Answers A. Lipid Metabolism and Glyoxylate Cycle 1. H H 3C C CH2 H H C C CH2 CH2 CH2 CH2 OH COO- CoA + ATP AMP + PPi H H 3C C CH2 H H C C CH2 CH2 CH2 CH2 CSCoA O OH 1 round beta oxidation H H 3C C CH2 H H C C CH2 CH2 O OH H H 3C C CSCoA CH2 H H C C First step beta oxidation H C OH C CSCoA H O NADPH + H+ H H 3C C NADP CH2 CH2 OH C C H H C CH2 H C OH CSCoA O H CH2 OH H 3C 2,4-dienoyl CoA reductase isomerase H H 3C C H2 + O CH2 C H2 C C CSCoA H O rest of 1 turn of beta oxidation, then 1 complete round CSCoA steps 3, 4 of beta oxidation Required: 2 ATP for activation 5 CoASH 4 NAD+ 1 NADPH 3 FAD Produces: 5 acetyl CoA 4 NADH 3 FADH2 1 NADP+ 2. Hydrolysis to glycerol + 3 caproic acid Yield for glycerol is 22 ATP Glycerol-3-P DHAP PYR Glycerol TCA -1 ATP 1 NADH 2 ATP/1 NADH NADH 3NADH/FADH2/GTP =3 ATP =5 ATP =3 ATP Total= 3 + 5 + 3 + 12 –1=22 Acetyl CoA =12 ATP 3 caproic acid 2 rounds of β−oxidation x 3 9 acetyl CoA TCA -6 ATP(to convert FFA to CoA ester) 12 + 18 ATP + 108 ATP 22 ATP from glycerol 24 ATP (β-oxidation) 108 ATP (TCA) 154 3. Arachidonic acid has 20 carbons, which yields 10 acetyl CoA that yield 120 ATP in the TCA cycle. To get the acetyl CoA, arachidonic acid goes through 9 turns of β−oxidation. A fully saturated C20 would give 9 NADH and 9 FADH2. Because this compound has 4 double bonds, 2 odd, 2 even, 7 FADH2 and 9 NADH are produced. Unsaturations at odd carbons require only an isomerase, bypassing the first FADH2 synthesizing oxidation step. Unsaturations at an even C require both the 2,4-dienoylCoA reductase and an isomerase. This consumes one NADPH. Total 120 + 14 + 27 = 161-2 for activation = 159 ATP 4. trait activated intermediates β-oxidation soluble acetyl CoA co-enzymes NAD+, FAD L-β-hydroxyacyl CoA mitochondria same as FA synthesis only two ATP to activate stereochemistry location intermediate structure ATP requirement fatty acid synthesis protein bound acetyl-ACP prot. bound malonyl-ACP NADPH D-β-hydroxyacyl CoA cytosol same as β-oxidation one ATP for every FA unit condensed 5. Animals can not catalyze the net conversion of acetyl-CoA to glucose because when they metabolize acetyl CoA to form the precursor for gluconeogenesis (malate that leaves the mitochondria to be oxidized to OAA and then OAA to be decarboxylated to PEP), two carbons are lost as CO2 in the two decarboxylating steps of the TCA cycle. Two carbons enter with acetyl CoA, but two carbons are lost in the conversion to malate. Therefore, if this malate is to be used to make glucose, something must be converted to OAA to continue the utilization of acetyl-CoA. This is pyruvate (a carbohydrate!) which is carboxylated via pyruvate carboxylase. Thus, no net gain of carbohydrate is achieved. 6. Although two carbons are lost as acetyl CoA goes through one turn of the TCA cycle, the carbons lost did not come from acetyl CoA. Therefore, labeled malate results, which can leave the mitochondria to undergo gluconeogenesis. 7. H 3C CH2 CH2 C S ACP -OOC CH2 C S ACP O O CO2 O H 3C CH2 CH2 C H C O COO- SACP H 3C CH2 CH2 C H2 C O NZ bound intermediate 8. The reaction is malate synthase. See the Handbook for the reaction. O SACP B. Amino Acid Metabolism 9. a. DOPA decarboxylase (makes DOPAMINE from DOPA), Histidine decarboxylase (makes histamine from histidine). b. alanine aminotransferase (same as alanine transaminase), aspartate aminotransferase (aspartate transaminase) 10. ASP must be catabolized to result in 15NH3. This is accomplished via an aminotransferase reaction using α-KG as the universal amino acceptor (aspartate: α-KG aminotransferase= asp transaminase) . 15N-GLU is then oxidatively deaminated, resulting in 15NH3 (GLU dehydrogenase or GLU:NAD+ oxidoreductase). This 15NH3 is used for carbamoyl-P synthesis (carbamoyl phosphate synthetase I). A second 15N-labeled ASP molecule is used in the urea cycle itself to condense with citrulline to produce argininosuccinate, that is converted to arginine and fumarate, then arginine is hydrolyzed to form urea and ornithine (don’t learn the names of the urea cycle enzymes). Hence, both nitrogens of urea will be labeled with 15N. 11. Pyridoxal Phosphate derives from pyridoxine. Tetrahydrofolate is derived from folic acid. 12. 2-O P O C NH 3 2 H H O + H COONH3 H COONH O NH2 + HPO42- 13. COOCH2 NADH + H+ H2O + NAD+ CH2 CH2 + NH3 CH2 H COO- O COO- COONH3 NH3 + HCO 3- + 2 ATP O 2- O3P O O 2- O3P O C NH2 C NH2 COO- + ASP CH2 H + 2 ADP + HPO42- + HPO42- COONH O NH2 14. Gluconeogenic means an amino acid breaks down into one or more compounds that can be used for the synthesis of glucose. Ketogenic means an amino acid breaks down into one or more compounds that cannot be used for the net synthesis of glucose, but can be used for energy production, or for the synthesis of ketone bodies or FA (for example two such compounds are acetyl CoA or acetoacetate). Some amino acids are both- that is part of the amino acid breaks down to a compound that can be used for net glucose synthesis and another part breaks down to acetyl CoA or a compound that breaks down to only acetyl coA. 15. GLU + NH3 + ATP GLN + ADP + Pi Common name: Gln synthetase CO2 + NH3 + 2ATP Carbamoyl-P + 2ADP + Pi Common name: Carbamoyl-P synthetase GLU + NAD+ + H2O ↔ αKG + NH3 + NADH H+ This can operate in the opposite direction as written (and as learned in class) under some conditions-when ammonia levels are high. Common name: Glutamate dehydrogenase For plants and microorganisms, all of the above reactions apply, plus the one below: 2GLU + NADP+ GLN + αKG + NADH + H+ Common name: Glutamate synthase (not present in animals) While this enzyme does not use free ammonia, it is part of the two enzyme cycle that results in net assimilation of NH3 into organic form (Glutamate synthetase and glutamate synthase). 16. They use all the same enzymes of the urea cycle except one to produce the amino acid arginine (the missing enzyme is arginase that cleaves arginine to ornithine and urea). 17. Methionine is the source of cysteine in animals. Therefore, if cysteine is supplied in the diet, then less methionine is required for this purpose and then the total dietary requirement for methionine is less. Homocysteine is an intermediate in cysteine biosynthesis in animals, therefore addition of homocysteine in the diet will also lower the dietary requirement for methionine. C. Regulation 18. a. glycogen synthesis would not be inhibited and glycogen breakdown would not be stimulated. Glycogen synthase would exist in the more active form and glycogen phosphorylase would exist in the b, or relatively inactive form. b. increase glycogen breakdown and slow down glycogen synthesis 19. a is the only correct answer. 20. The temporal order is d, b, a, c 21. Caffeine would inhibit the hydrolysis of cAMP to AMP and prolong the stimulation of glycogen breakdown and the inhibition of glycogen synthesis initiated by adrenaline. 22. The different products of epinephrine-stimulated glycogen breakdown in muscle and liver reflect the different roles that these two organs play in carbohydrate metabolism in mammals. The liver supplies glucose to other organs of the body, hence glycolysis would not be stimulated, the Glc-1-P produced is isomerized and dephosphorylated in order to leave the cell. In contrast, Glc-1-P made in the muscle is isomerized to Glc-6-P and used there for ATP production via glycolysis.