Revised 9/17/03 1 CHEMISTRY 153C Study Questions and Problems with Answers II Winter 2001 1. Compare a reaction selected from the -oxidation cycle of fatty acid oxidation or the biosynthesis of acetoacetate with a closely analogous reaction of the tricarboxylic acid cycle. Select reactions for comparison in which the chemical changes involved and the mechanistic features bear a close resemblance to one another. Illustrate the reactions and comment on the most important features they have in common. 2. Illustrate with structural formulas one reaction of physiological importance for each of the following cases. Coenzymes may be abbreviated in standard fashion; intermediates and mechanisms need not be indicated. In each case state briefly a function in metabolism for the reaction you have shown. a) A reaction catalyzed by pyrophosphatase. 1. One example: oxidation:RCH2CH2CH2C SCoA FAD+R- CH2HC = CH CSCoAO+ FADH2TCA cycle: OCCH2CH2CO+FADOOCHC = CHCOO+ FADH2OOO Both reactions are dehydrogenases which catalyse the removal of hydrogens from and carbons adjacent to a carboxylate or carboxylic acid derivative to generate a trans-unsaturated (E) product. Both enzymes are flavoproteins in which the hydrogens (2 H+ + e) are transferred to FAD as an acceptor. Other possibilities include: 1) EnoylCoA hydratase/fumarase (or possibly cis-aconitase). 2) L-3-hydroxyacyl CoA dehydrogenase/L-malate dehydrogenase. 3) HMGCoA synthase/citrate synthase.Revised 9/17/03 2 2. b) A reaction in which carnitine participates as a substrate. 2. (a) OPOPOHH2O2 OPOH O+OOOOO This reaction serves to keep the inorganic pyrophosphate concentrations low and thus make reactions which generate pyrophosphate as a product (such as fatty acid activation) more favorable thermodynamically. 2. (b) CH3(CH2)nC SCoA + HO CHCH2COOOCH2CH3 NCH3CH3CH3(CH2)nCOCHCH2COOOCH2CH3 NCH3CH3CoA-SH+ ++ This reaction (catalyzed by carnitine: fatty acyl trasferase) participates in the transport of long chain fatty acylCoA accross the mitochondrial inner membrance and regeneration in the matrix to serve as a substrate for fatty acid oxidation.Revised 9/17/03 3 2. c) A reaction catalyzed by malate synthase. 3. Unless otherwise indicated, use structural formulas for reactants and products except for coenzymes which can be abbreviated in standard fashion: a) Illustrate a reaction that is catalyzed by heart cells but not liver cells and is responsible for the ability of heart cells to use ketone bodies as an energy source. b) Would an auxiliary enzyme (or enzymes) be required to effect the complete oxidation of the activated fatty acid shown below to acetylCoA by -oxidation? CH3(CH2)5CH (CH2)10OHCOSCoA 2. (c) COOCH2OCH3CSCoACoASHCOOCHO HCH2COO+OOH This reaction serves as part of the glyoxylate pathway, the function of which is to provide for a net synthesis of dicarboxylic acids from acetylCoA for replenishment (anaplerosis) and biosynthesis. 3. (a) OOCCH2CH2C SCoA +OOCCH2CCH3OOCCH2CH2 COO+CoA S CCH2CCH3OOOORevised 9/17/03 4 c) Show the structure of 3-hydroxy-3-methylglutarylCoA and indicate clearly with an asterisk the labeling pattern expected in this substance when it is produced in liver mitochondria that are carrying out the oxidation of 2-[14C]-palmitoylCoA. CH3CH2(CH2CH2)6CH2CSCoAO* 4. The cleavage of acetoacetylCoA to 2 acetylCoA catalyzed by the enzyme 3-ketothiolase is thermodynamically favorable under standard state conditions ( Go = -6.7 kcal mol-1). If this is the case, how could the proposed synthesis of acetoacetylCoA from acetylCoA in a reaction catalyzed by 3-ketothiolase serve as a step in the biosynthesis of ketone bodies in liver mitochondria? Be as specific as you can in describing factors that may be involved. 3. (b) Yes. Five rounds of -oxidation would yield a 2-hydroxyacyl-CoA that is not a normal intermediate of -oxidation and would presumably require an auxiliary enzyme or enzymes to convert it to a normal intermediate. 3. (c) OSCoAC*CH2C*CH2COO*CH3 OH 4. During ketogenesis, the active condensation of acetoacetylCOA with acetylCoA catalyzed by HMGCoA synthase is very favorable and will keep the concentration of acetoacetylCoA low relative to acetylCoA. Even under standard state conditions the coupled process will be favorable. ~ G cal/mol 2 acetylCoA acetoacetylCoA + CoASH +6.7 acetoacetylCoA + acetylCoA + H2O HMGCoA + CoASH - 7.5 ______________________________________________________________________________ 3 acetylCoA + H2O HMGCoA + 2CoASH - 0.8 It will be even more favorable if HMGCoA is being cleaved and ketone bodies are transported out of the matrix. It may also be driven under cellular conditions by high acetyl-CoA.Revised 9/17/03 55. For some years after the general features of the process of -oxidation of fatty acids to acetylCoA had been elucidated, it was assumed by many biochemists that fatty acid biosynthesis from acetylCoA would be accomplished by the reversal of the -oxidation pathway. However, experimental efforts to demonstrate this were fruitless. What features of the -oxidation pathway would appear to make it a poor candidate as the scheme for the biosynthesis of fatty acids when operating in the reverse direction? 6. Illustrate how the following transformation may occur in a bacterial culture growing on acetate as a sole carbon source. More than one enzyme-catalyzed reaction may be involved. Note that this is not a balanced equation. Illustrate reactions in sequential fashion, using structures for reactants and products except for coenzymes, which may be abbreviated in standard fashion. Mechanisms are not required. CH3COSCoAHCCH2COO-CCOO-HO HCOO-HO C HCOO-CH2COO-COO-CH2CH2COO-++ 5. It has been found that metabolic pathways are characterized by large negative G values under physiological conditions and thus are essentially unidirectional. Therefore, it would not be anticipated that -oxidation and synthesis would occur by reversal of the same pathway since it would need to be poised somewhere near equilibrium for that to occur. Since -oxidation occurs in the mitochondrion where the concentration of NADH is kept low by its strongly