Biochemistry 3300 Problems (and Solutions) Metabolism I

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1 (1) Provide a reasonable systematic name for an enzyme that catalyzes the following reaction: fructose + ATP > fructose-1 phosphate + ADP (2) The IUBMB has a developed a set of rules for classifying enzymes based upon the type of reaction they catalyze. The classification scheme assigns an Enzyme Commission (EC) number to each enzyme. Answer the following questions regarding EC numbers and the class of reaction catalyzed: (a) What name is associated with class 4 enzymes and what type of reaction do they catalyze? (b) Oxidoreductases catalyze redox reactions. What class of enzymes are oxidoreductases? (c) What types of reactions are catalyzed by ligases? (3) List four characteristics that define a metabolic pathway. (4) Identify two amino acids that commonly act as nucleophiles in enzyme catalyzed reactions. (5) What is the difference between an (1) isomerization reaction and (2) a rearrangement? (6) Enzyme catalyzed reactions that make or break C-C bonds typically involve carbanion intermediates. One means of stabilizing a carbanion intermediate involves the formation of a Schiff's base. Draw a strcuture of : (a) a Schiff's base carbanion (b) the resonance stabilized Schiff's base in the eneamine form (7) How can metabolic inhibitors be used to determine the sequence of reactions in a metabolic pathway? (8) Isotopes of C, N, S, P and H have been extensively used to investigate metabolic pathways. Why? (9) The standard transformed free energy change for the following bimolecular reaction: Glucose + ATP ----> Glucose-6-phosphate + ADP is kj/mol. Consequently, this reaction is spontaneous under standard conditions. Under what conditions would this reaction NOT be spontaneous at 298K? (10) If an enzyme catalyzed the direct transfer of a phosphate to glucose Glucose + P i ---> Glucose-6-phosphate the standard, transformed free energy change would be kj/mol. Under what conditions would this reaction be favourable at 298K?

2 (11) Place an 'x' on each of the high energy bonds shown in the structures below. (12) Consider the oxidation-reduction reaction between cytochrome b and ubiquinol. (a) What is the G'º for the reduction of cytochrome b by ubiquinol? (Note: you will need to use the tables of standard reduction potentials) (b) Under what conditions will the reduction of cytochrome b by ubiquinol be favourable at 298K? (13) Indicate whether the following statements are true or false. If false, change a single word or number to make the statement true. (a) (6-13 C)Glucose (ie. C6 label) is converted to (1-13 C)Glyceraldehyde-3-phosphate during the preparatory phase of glycolysis. (b) Phosphoglycerate kinase requires catalytic amounts of 2,3-bisphosphoglycerate for activity. (c) Glyceraldehyde-3-phosphate dehydrogenase couples the addition of phosphate to glyceraldehyde-3-phosphate and the reduction of NAD +. (14) Identify the glycolytic enzyme with the following properties: (a) Forms a Schiff's base intermediate during its catalytic cycle. (b) Synthesized in an inactive form. (c) Is inhibited by alkylating agents such as iodoacetate. (d) Is a lyase. (15) Answer the following questions regarding glycolysis: (a) What is the overall chemical reaction for the 'preparatory' phase of glycolysis? (b) What is substrate-level phosphorylation? (c) Under cellular conditions, which glycolytic enzymes are likely to be regulatory targets? (d) Which glycolytic intermediates are 'high-energy' compounds?

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4 Answers 1- Fructose:ATP phosphotransferase or Fructose:ATP kinase 2- (a) Lyases catalyze group eliminations that form double bonds (b) Class 1 (c) Bond formation coupled to ATP hydrolysis. 3- Metabolic pathways are (1) irreversible, (2) have a committed step, (3) are regulated and (4) catabolic and anabolic pathways differ. 4- Serine, threonine and cysteine are nucleophilic in their ionized form, while histidine and lysine are nucleophiles in their neutral-charged form. 5- Isomerization reactions inevitably involve the movement of an H atom without changing the carbon backbone while rearrangements involve changes to the carbon backbone. 6-(a) Schiff's base carbanion (b) Schiff's base eneamine form 7- Metabolic inhibitors specifically block a metabolic pathway and result in the accumulation of metabolic pathway intermediates. By identifying the metabolic pathway intermediate(s) that accumulate, one can identify the step in a metabolic pathway that has been inhibited. The use of several metabolic inhibitors that act at different points in the pathway (together with 'chemical intuition') is sufficient to propose and verify the sequence of reactions associated with a metabolic pathway. 8- Greatest difficulty in metabolism research is identifying the intermediates of a metabolic pathway. The large number of related compounds in an organism make the identification of metabolic intermediates difficult. Further, many metabolic intermediate are only present at very low levels making them difficult to detect. The use of isotopes (especially radioisotopes) can overcome this problem as they can be readily detected at very low concentrations and serve to 'tag' or 'label' each of the metabolic intermediates of a pathway. 9 - Reactions are only spontaneous when the G' < 0. Given our equation for calculating G': G' = G' + RT ln ([Products]/[Reactants]) 0 > G' + RT ln ([Products]/[Reactants]) for a spontaneous reaction 0 > kj mol J mol -1 K K ln ([Products]/[Reactants]) 16.7 kj mol -1 / 2.48 kj mol -1 > ln ([Products]/[Reactants])

5 6.73 > ln ([Products]/[Reactants]) e 6.73 > ([Products]/[Reactants]) > ([Products]/[Reactants]) When the ratio of Products to Reactants is greater than or equal to 837.1, the reaction will no longer be spontaneous. (Note, at exactly fold excess of products to reactants the reaction will be at equilibrium) 10 - This reaction will only be favourable when G' is negative. G' = G' + RT ln ([Products]/[Reactants]) 0 > G' + RT ln ([Products]/[Reactants]) for a spontaneous reaction 0 > 13.8 kj mol J mol -1 K K ln ([Products]/[Reactants]) kj mol -1 / 2.48 kj mol -1 > ln ([Products]/[Reactants]) > ln ([Products]/[Reactants]) e > ([Products]/[Reactants]) > ([Products]/[Reactants]) or < ([Reactants]/[Products]) While the reactant concentration is fold higher (or more) than the product concentration, the reaction will be favourable Please note that in the case of PEP (left) and 1,3-bisphosphoglycerate (top-middle) you could have placed the 'x' on the adjacent anhydride-like bond. 12- (a) Cyto b (ox) + e - cyto b (red) 0.077V ubiquinol + H 2 ubiquinone + 2e - + 2H V 2 Cyto b (ox) + ubiquinol + H 2 2 cyto b (red) + 2H V G'º = -nf E'º = -2 (96.5 kj/v mol) (0.032 V mol) = -6.2 kj/mol (b) There are two ways to solve this problem; using standard reduction potentials or using free energy changes.

6 G' = G' + RT ln ([Products]/[Reactants]) 0 > G' + RT ln ([Products]/[Reactants]) for a spontaneous reaction 0 > -6.2 kj mol J mol -1 K K ln ([Products]/[Reactants]) -6.2 kj mol -1 /2.48 kj mol -1 > ln ([Products]/[Reactants]) > ([Products]/[Reactants]) or 12.1[Reactants] > [Products] The reaction will no longer be favourable when the products are 12.1 times more concentrated than reactants. OR E' = E ' - (RT/nF) ln ([Products]/[Reactants]) 0 < E ' - (RT/nF) ln ([Products]/[Reactants]) for a spontaneous reaction 0 < V - (8.31 J mol -1 K K / (2) 96,480 J V -1 mol -1 ) ln ([Products]/[Reactants]) 0 < V ( V) ln ([Products]/[Reactants]) V/ V > ln ([Products]/[Reactants]) 12.1 > ([Products]/[Reactants]) or 12.1[Reactants] > [Products] The reaction will no longer be favourable when the products are 12.1 times more concentrated than reactants. 13- (a) False. Change 1 to 3. (b) False. Change kinase to mutase. (c) True. 14- (a) Aldolase catalyzes the cleavage of a C-C bond via a Schiff's base. (b) Phosphoglycerate Mutase must be activated by 2,3-bisphosphoglycerate. (c) Glyceraldehyde-3-phosphate dehydrogenase has an active site thiol. (d) Enolase forms a double bond while eliminating a H 2 O molecule Aldolase forms a ketone while eliminating glyceraldehyde-3-phosphate. 15- (a) Glucose + 2ATP 2 Glyceraldehyde-3-phosphate + 2ADP (b) P i is a substrate in the formation of a 'high-energy' phosphodiester. In glycolysis, glyceraldehyde-3-phosphate, P i and NAD + are converted to 1,3-bisphosphoglycerate and NADH + H +. Phosphate addition to the aldehyde of glyceraldehyde-3-phosphate generates a 'high-energy' phosphodiester bond in 1,3-bisphosphoglycerate. (c) Regulatory targets are typically enzymes that catalyze reactions with large G'. In the particular case of glycolysis, only hexokinase, phosphofructokinase and pyruvate kinase have large G's and are regulatory targets. Aldolase (unfavourable under standard conditions) and phosphoglycerate kinase (Energy coupled with glyceraldehyde-3-phosphate Dehydrogenase) have much smaller G's under cellular conditions and are not regulatory targets. (d) 1,3-bisphosphoglycerate, phosphoenolpyruvate