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- 1. (a) The reaction catalyzed by citrate synthase is the first step of the TCA cycle. In glycolysis, two key reactions to produce ATP occur because an unfavorable reaction is coupled to another reaction that is thermodynamically favorable. The reaction catalyzed by citrate synthase is similarly coupled to an unfavorable reaction in the TCA cycle. Write the unfavorable TCA reaction using structural formulas and write the key step that drives the two coupled reactions forward. What is the overall AG" of the coupled reactions? (b). K(yM) 25.7 Inhibitor Bromoacetyl-CoA ATP NADH 6800 8300 The inhibitor constants for three inhibitors of por- cine citrate synthase are summarized in the table on the right. The compounds were all determined to bind in the ac- tive site as competitive inhibitors of acetyl-CoA. Because they bind as competitive inhibitors, all three inhibitors must exhibit structural similarity to some part of acetyl-CoA. Look up in the textbook the structural formulas for…2. Please, determine the answers of these multiple choices, they’re in a,b,c’s. A) The role of oxygen in oxidative phosphorylation (cellular breathing) is: -To be the last electron acceptor in the electron transfer chain -To hydrolyse carbohydrates - To add hydrogen ions to pyruvic acid at the end of glycolysis -To provide electrons for NADP reduction -To provide hydrogen ions B) Which molecule has the most potential energy? -glucose -phosphate -fructose 1-6 diphosphate -ethanol -I'ATP C) What is the product of fermentation in yeasts? -carbonic acid -oxygen -ATP -lactic acid -ethanol2B. Calculate the total number of ATP that will be generated from the complete B-oxidation of oncobic acid (CH:(CH,),CH=CH(CH,),COOH; 15:1 cis-A9) in an organism that has all the standard required enzymes for metabolism but which pumps only 2 H" in complex I, 0 H' in complex II, 2 H* in complex III and 4 H' in complex IV. The F,F, ATPase is composed of 3 a/ß subunits and 10 c subunits. Show all of your calculations. HO
- 2. Prokariotic aldolase are mediated by a Zn2+ and are inhibited by EDTA, eukaryotic aldolases though are inactivated by sodium borohydride. A) Draw out the mechanism of the eukaryotic aldolase and propose a parallel mechanism for the zinc catalyzed prokaryotic reaction. B) Propose a strategy that could be could be used to specifically inhibit prokaryotic aldolases and not eukaryotic?2. The mechanism of HMG-CoA reducatse enzyme activity involves several stages. For the catalytic reaction to proceed, another substrate known as NADPH, which acts as a reducing agent is required. The kinetics of enzyme activity with NADPH at non-limiting amounts of HMG-CoA in the absence or presence of compactin is shown below. a) Based on the description above, which of the six types of enzymes does HMG-CoA reductase belong to? b) What is the Km for the reaction with no inhibitor present? Do NOT forget the units. Answers can be expressed either as a fraction or in decimals. c) What happens the measured Km with increasing amounts of compactin? (select one) increases decreases does not change d) What happens to the measured Vmax with increasing amounts of compactin? (select one) increases decreases does not change e) Based on your answers above, what type of inhibitor is compactin relative to its effects on enzyme activity NADPH? with -60 -40 1/V, (nanomoles/min)-1 -20 0.2 0.18 0.16…10. Consider the beta oxidation of stearic acid (C18:0): How many ATP are generated in complete oxidation of stearic acid? How many NADH are generated in complete oxidation of stearic acid? How many FADH2 are generated in complete oxidation of stearic acid?
- 2. Formation of OAA in mitochondria. In the last reaction of the TCA cycle, malate is dehydrogenated to regenerate the OAA necessary for the entry of acetyl-coA into the cycle: L-Malate + NAD+ → OAA + NADH + H+ AGo = 30.0 kJ/mol (a) Calculate the equilibrium ponstant for this reaction at 25 C. b) Because AGo assumes a standard pH of 7, the equilibrium constant calculated in (a) corresponds to K'eg _JOAA][NADH] [L-malate][NAD*] The measured concentration of L-malate in rat liver mitochondria is about 0.2 mM when the ratio [NAD*] / [NADH] is 10. Calculate the concentration of OAA at pH 7 in these mitochondria. (c) To appreciate the magnitude of the mitochondrial OAA concentration, calculate the number of OAA molecules in a single rat liver mitochondrion. Assume the mitochondrion is a sphere of diameter 2.0 µm. (From problem 10, chapter 16)1. Cyanide, oligomycin, and 2,4-dinitrophenol are all inhibitors of oxidative phosphorylation in mitochon- dria. Provide an explanation to the following conditions regarding these potent inhibitors. (a) Explain why adding cyanide to an active in vitro suspension of mitochondria blocks ATP synthesis. What happens to the rate of ATP synthesis when 2,4-dinitrophenol is added to this mitochon- drial suspension after it was treated with cyanide? (b) Explain why the rate of oxygen consumption decreases in an in vitro suspension of mito- chondria when oligomycin is added. What happens to the rate of oxygen consumption in this oligomycin- inhibited system after adding 2,4-dinitrophenol? Explain.14. Inhibitors. Rotenone inhibits electron flow through NADH-Q oxidoreductase. Antimycin A blocks electron flow between cytochromes b and c₁. Cyanide blocks elec- tron flow through cytochrome oxidase to O₂. Predict the relative oxidation-reduction state of each of the following respiratory-chain components in mitochondria that are treated with each of the inhibitors: (a) NAD+ (b) NADH-Q oxidoreductase (c) coenzyme Q (d) cytochrome c₁ (e) cytochrome c (f) cytochrome a
- 2. Regarding the glycolysis metabolic pathways covered in class: A. In the EMP metabolic pathway, what chemical is produced by anabolism of pyruvate? (1 step away) B. There are two products of step 4 in the EMP pathway. If they were not phosphorylated, how would you describe these as monosaccharides (for example, an aldohexose and a ketopentose)? C. In the TCA Cycle, what chemical is produced by catabolism of isocitrate? (1 step away) D. Describe the major differences between primary, secondary and partial oxidation metabolism in terms of (i) cell growth rate, (ii) oxygen consumption rate, and (iii) products formed.1. The first step in the payoff phase of glycolysis is catalyzed by the enzyme glyceraldehyde 3-phosphate dehydrogenase, an enzyme that contains a nucleophilic cysteine playing a central role in the reaction. A) In the direction of gluconeogenesis, what reaction does this enzyme catalyze? AG° = -6.3 kcal/mol for this reaction in the direction of gluconeogenesis. Based on what you know about the substrates involved, provide two chemical reasons as to why the AGO of this reaction is negative.1. What are the three major pathways that eventually become entry points of molecules into the Krebs Cycle? Which molecules from these respective pathways are integrated into the Krebs Cycle? 2. How is a "committed step" defined in the context of a metabolic pathway and why are they important? Which steps and/or enzymes are involved in the committed steps in the Krebs Cycle? What are the possible implications of these steps were deregulated? 3. What are the three Krebs Cycle inborn errors mentioned in the paper? Why are they called deficiencies? What do you think are the probable causes for these?