LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL. 2. BIOENERGETICS AND METABOLISM - 2014

PART II. BIOENERGETICS AND METABOLISM

16. THE CITRIC ACID CYCLE

Questions and Problems

1. Stoichiometry of The Citric Acid Cycle.

Eight Enzymes are involved in The Citric Acid cycle: citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase.

a) Write the chemical equation for the reaction catalyzed by each enzyme.

b) Name the Cofactors required for the action of each enzyme.

c) For each enzyme, identify the type of reaction it catalyzes: Condensation (carbon-carbon bond formation), dehydration (Water elimination), Hydration (water addition), decarboxylation (CO₂ elimination), oxidation-reduction, substrate-level phosphorylation, or isomerization.

d) Write the overall equation for The conversion of acetyl-CoA to CO₂.

2. Overall equations of Glycolysis and the citric acid cycle.

Write the complete biochemical equations for glucose METABOLISM via glycolysis and for the citric acid cycle, accounting for all cofactors.

3. Identifying oxidation-reduction reactions in metabolic pathways.

The biochemical strategy of many living organisms involves the gradual oxidation of Organic compounds to CO₂ and H₂O, capturing a major portion of the released energy in the form of ATP. It is important to be able to recognize the oxidation-reduction stages of metabolism. The reduction of an organic molecule occurs via hydrogenation across a double bond (Equation 1) or across a single bond accompanied by its Cleavage (Equation 2). Conversely, oxidation results from dehydrogenation. In biochemical oxidation-reduction (redox) reactions, the Coenzymes NAD and FAD function as hydrogen carriers in the presence of their respective enzymes.

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For each of the conversions listed below, indicate whether oxidation or reduction takes place. Write a balanced equation for each redox reaction by adding H-H and, where necessary, H₂O.

4. Relationship between the energy yield of a reaction and the oxidation state of carbon.

Eukaryotic Cells can use glucose (C₆H₁₂O₆) and hexanoic (caproic) acid (C₆H₁₄O₂) for cellular Respiration. Using the structural formulas of these compounds, determine which of them yields more energy per gram upon complete oxidation (to CO₂ and H₂O).

5. Nicotinamide coenzymes as hydrogen carriers in oxidation-reduction reactions.

In the presence of the appropriate dehydrogenase, nicotinamide coenzymes (see Fig. 13-24) can participate in reversible oxidation-reduction reactions with specific substrates. In such reactions, NADH + H⁺ serves as a hydrogen source (see Problem 2). When the coenzyme is oxidized, the substrate is invariably reduced:

For each of the reactions given below, determine whether the substrate was oxidized, reduced, or its oxidation state remained unchanged (see Problem 2). If the oxidation state of the substrate changed, write the equation by adding the necessary amounts of NAD⁺, NADH, H⁺, and H₂O. The purpose of this exercise is to determine in which reactions a redox coenzyme must participate.

6. Cofactors and mechanism of the reaction catalyzed by Pyruvate dehydrogenase.

Describe The Role of each cofactor involved in the reaction catalyzed by the pyruvate dehydrogenase complex.

7. Thiamine deficiency in the body.

An individual with an inadequate Dietary intake of thiamine exhibits high levels of pyruvate in the Blood. Explain the cause.

8. The reaction catalyzed by isocitrate dehydrogenase.

What type of chemical reaction takes place during the conversion of isocitrate to α-ketoglutarate? Name all the cofactors involved in this reaction and explain their role. Which other reaction(s) in the citric acid cycle belong to the same type?

9. Stimulation of oxygen consumption by oxaloacetate and malate.

In the early 1930s, Albert Szent-Györgyi reported an interesting observation: The addition of a small amount of oxaloacetate or malate to a suspension of minced pigeon breast Muscle enhanced oxygen consumption by the preparation. Surprisingly, The amount of oxygen consumed was 7 times greater than required for the Complete oxidation of the added oxaloacetate or malate (to СO₂ and Н₂O). Why does the addition of oxaloacetate and malate stimulate oxygen consumption? Why does the amount of oxygen consumed vastly exceed that required for the complete oxidation of the added oxaloacetate or malate?

10. Formation of oxaloacetate in Cell/35.html">Mitochondria.

The final stage of the citric acid cycle involves the dehydrogenation of malate, which regenerates oxaloacetate required for The entry of a new acetyl-CoA molecule into the cycle:

L-Malate + NАD⁺ —> oxaloacetate + NАDН + Н⁺

∆G′° = 30 kJ/mol

a) Determine the Equilibrium Constant of the reaction at 25 °C.

b) Since ∆G′° corresponds to the standard value at pH 7, the equilibrium constant (from part a) is determined by the formula:

Measurements show that the concentration of L-malate in rat Liver mitochondria is -0.2 mM, and [NАD⁺]/[NАDН] = 10. Determine the concentration of oxaloacetate in rat liver mitochondria at pH 7.

c) To estimate the concentration of oxaloacetate in mitochondria, calculate the number of oxaloacetate molecules in a single mitochondrion. Assume the mitochondrion is a spherical particle with a diameter of 2 µm.

11. Cofactors in the citric acid cycle.

Suppose a mitochondrial extract is prepared containing all soluble matrix enzymes, but dialysis has resulted in the loss of all low-molecular-weight cofactors. What must be added to the extract for the conversion of acetyl-CoA to СO₂ to proceed?

12. Riboflavin deficiency in the body.

How will riboflavin deficiency affect the functioning of the citric acid cycle? Explain your answer.

13. Oxaloacetate levels.

What factors can decrease the amount of oxaloacetate entering the citric acid cycle? How can the pool of oxaloacetate be replenished?

14. Energy yield of the citric acid cycle.

The reaction catalyzed by succinyl-CoA synthetase leads to The formation of the energy-rich compound GTP. How is the Free energy stored in GTP "coupled" with the cellular energy reserves embodied in ATP?

15. Study of Cellular respiration in isolated mitochondria.

Cellular respiration can be studied in isolated mitochondria by measuring The rate of oxygen consumption under various conditions. If a 0.01 M sodium malate solution is added to actively respiring mitochondria using pyruvate as an energy source, respiration rapidly ceases and one of the metabolic intermediates begins to accumulate.

a) What is the Chemical Structure of this intermediate?

b) Explain why it accumulates.

c) Explain why oxygen consumption stops.

d) Aside from directly removing malate, what other method can be used to relieve the inhibition it causes? Explain your answer.

16. Experiments with labeled compounds in isolated mitochondria.

Metabolic pathways of organic compounds are frequently investigated using radiolabeled substrates to trace The Fate of the label.

a) How can one verify that glucose added to a suspension of isolated mitochondria is converted into CO₂ and H₂O?

b) [3-¹⁴C]pyruvate (labeled at the methyl group) was added as a pulse to mitochondria. What position will the labeled carbon occupy after one turn of the citric acid cycle? Explain your answer by tracing the path of the label throughout the cycle. After how many turns of the cycle will all of the labeled pyruvate be released as CO₂?

17. The fate of CO₂ during Gluconeogenesis.

In the first bypass of gluconeogenesis—the conversion of pyruvate to phosphoenolpyruvate (PEP)—pyruvate is carboxylated by pyruvate carboxylase to form oxaloacetate, which is subsequently decarboxylated to PEP by PEP carboxykinase (Ch. 14). Because the addition of CO₂ is immediately followed by its release, one might expect that ¹⁴C from ¹⁴CO₂ would not be incorporated into PEP, glucose, and other gluconeogenic intermediates. However, it has been found that during glucose synthesis in rat liver in the presence of ¹⁴CO₂, ¹⁴C gradually appears in PEP and subsequently at the C-3 and C-4 positions of the glucose molecule. How does the ¹⁴C label find its way into PEP and glucose? (Hint: During gluconeogenesis in the presence of ¹⁴CO₂, some four-carbon Intermediates of the citric acid cycle also become labeled.)

18. Catabolism of [1-¹⁴C]glucose. An actively respiring bacterial culture was incubated briefly with [3-¹⁴C]glucose, after which the intermediates of the citric acid cycle and glycolysis were isolated. Indicate THE POSITION OF the label in the compounds listed below. Consider only the label incorporated During the first passage of the labeled glucose through the metabolic pathways.

a) Fructose-1,6-bisphosphate.

b) Glyceraldehyde-3-phosphate.

c) Phosphoenolpyruvate.

d) Acetyl-CoA.

e) Citrate.

f) α-Ketoglutarate.

g) Oxaloacetate.

19. The Biological Role of thiamine.

In beriberi, a disease caused by thiamine deficiency, patients exhibit elevated blood levels of pyruvate and α-ketoglutarate, particularly after consuming a glucose-rich meal. How is this related to thiamine deficiency?

20. Synthesis of oxaloacetate in the citric acid cycle.

Oxaloacetate is formed in the final step of the citric acid cycle via the NAD⁺-dependent oxidation of L-malate. Is the synthesis of oxaloacetate from acetyl-CoA possible using only the enzymes and cofactors of the citric acid cycle without depleting cycle intermediates? Explain your answer. How are the pools of oxaloacetate withdrawn from the cycle for biosynthetic reactions replenished?

21. Depletion of oxaloacetate pools.

In mammalian liver, oxaloacetate can serve as a Starting Material for gluconeogenesis (Ch. 14). Will the intensive use of oxaloacetate for gluconeogenesis affect the operation of the citric acid cycle? Explain your answer.

22. MECHANISM OF ACTION of fluoroacetate, used as a rodenticide.

Fluoroacetate is used to control rodents and is therefore produced industrially; this compound also occurs naturally in a South African plant. Upon entering The Cell, fluoroacetate is converted to fluoroacetyl-CoA in a reaction catalyzed by acetate thiokinase:

The Toxic Effect of fluoroacetate was studied in isolated rat hearts. Following Heart perfusion with a 0.22 mM fluoroacetate solution, glucose uptake decreased, the rate of glycolysis dropped, and glucose 6-phosphate and fructose 6-phosphate began to accumulate. Determination of citric acid cycle intermediates showed that their concentrations were below normal, whereas only the citrate concentration exceeded normal by 10-fold.

a) At what stage does the citric acid cycle halt? Why does citrate accumulate while the pools of other cycle intermediates are depleted?

b) In the citric acid cycle, fluoroacetyl-CoA undergoes enzymatic transformations. What is The structure of the end product of fluoroacetate metabolism? Why does it block the citric acid cycle? How can this inhibition be reversed?

c) Why did glucose consumption and glycolysis decrease after heart perfusion? Why did hexose monophosphates accumulate?

d) Why is fluoroacetate poisoning fatal?

23. Formation of L-malate in winemaking.

The tart taste of certain wines is associated with a high concentration of L-malate. Write The sequence of reactions by which Yeast forms L-malate from glucose under anaerobic conditions in the presence of dissolved CO₂ (HCO₃). Note that the overall Fermentation reaction should not involve the consumption of nicotinamide coenzymes or citric acid cycle intermediates.

24. Synthesis of α-ketoglutarate.

α-Ketoglutarate plays a crucial role in The Biosynthesis of several Amino Acids. Write the sequence of enzymatic reactions by which α-ketoglutarate is formed from pyruvate. The proposed reaction pathway should not result in the net consumption of citric acid cycle intermediates. Write the equation for the overall reaction and indicate the source of each reactant.

25. Amphibolic pathways.

Explain with Examples what is meant by the statement that the citric acid cycle is an amphibolic pathway.

26. Regulation of pyruvate dehydrogenase complex activity.

In animal Tissues, the rate of pyruvate conversion to acetyl-CoA is regulated by the ratio between the active (phosphorylated) and inactive (non-phospho-

rylated) PDH complexes. How is the rate of this reaction affected by treating a rabbit muscle mitochondrial preparation containing the PDH complex with (a) pyruvate dehydrogenase kinase, ATP, and NADH; (b) pyruvate dehydrogenase phosphatase and Ca²⁺; (c) malonate.

27. Industrial synthesis of citric acid.

Citric acid is used to impart flavor to soft drinks and many other food products. The global market for citric acid production is estimated at hundreds of millions of dollars per year. Industrial production employs the mold fungus Aspergillus niger, which is cultivated on sucrose under strictly controlled conditions.

a) The yield of citric acid strongly depends on the FeCl₃ concentration in the nutrient medium (see graph). Why does the citric acid yield decrease when the Fe³⁺ concentration is either above or below its optimal value of 0.5 mg/L?

b) Propose a reaction sequence by which A. niger produces citric acid from sucrose. Write the equation for the overall reaction.

c) Is it necessary to aerate the nutrient medium during fungal cultivation—that is, is this process aerobic or anaerobic? Explain your answer.

28. Regulation of citrate synthase activity.

In the presence of a saturating concentration of oxaloacetate, the dependence of citrate synthase activity from pig heart tissue on the acetyl-CoA concentration exhibits a sigmoidal profile (see graph). Upon addition of succinyl-CoA, the curve shifts to the right, and the sigmoidal Nature of the dependence becomes more pronounced.

Based on these observations, propose a mechanism by which succinyl-CoA regulates citrate synthase activity. (Hint: See Fig. 6-29, vol. 1.) Why is succinyl-CoA an appropriate signal for the Regulation of the citric acid cycle? How does The regulation of citrate synthase control the rate of cellular respiration in pig heart tissue?

29. Regulation of pyruvate carboxylase.

The carboxylation of pyruvate catalyzed by the enzyme pyruvate carboxylase proceeds at an extremely low rate in the absence of the allosteric activator acetyl-CoA. Explain how this regulatory feature relates to the observation that following a meal rich in Fatty acids (triacylglycerols) and poor in CARBOHYDRATES (glucose), The oxidation of glucose to CO₂ and H₂O decreases, whereas the oxidation of acetyl-CoA derived from fatty acids is enhanced.

30. Link between cellular respiration and the citric acid cycle.

Although oxygen does not directly participate in the citric acid cycle, the cycle operates exclusively in the presence of oxygen. Why?

31. Influence of the [NADH]/[NAD⁺] ratio on the citric acid cycle.

How will the citric acid cycle respond to a rapid increase in the [NADH]/[NAD⁺] ratio within the mitochondrial matrix? Explain your answer.

32. Thermodynamics of the citrate synthase reaction in cells.

Citrate is formed by the condensation of acetyl-CoA with oxaloacetate, catalyzed by citrate synthase:

Oxaloacetate + acetyl-CoA + H₂O —> citrate + CoA + H⁺

In rat heart mitochondria at pH 7.0 and 25 °C, the concentrations of reactants and products were determined as follows (μM): oxaloacetate 1, acetyl-CoA 1, citrate 220, CoA 65. The standard free-energy change for this reaction is -32.2 kJ/mol. What is the direction of the metabolic flux for this reaction in rat heart cells? Explain your answer.

33. Reactions of the pyruvate dehydrogenase complex.

The two stages of Oxidative Decarboxylation of pyruvate (steps (4) and (5) in Fig. 16-6) do not involve any of the three carbon atoms of the pyruvate molecule, yet they are essential for the functioning of the PDH complex. Explain this fact.

34. Mutations in the citric acid cycle.

Many human diseases are associated with the loss of specific enzymatic activities resulting from genetic mutations. However, the loss of activity of any enzyme participating in the citric acid cycle is extremely rare. How can this be explained?

35. The citric acid cycle and The Glyoxylate cycle.

In organisms (such as E. coli) that possess both the citric acid cycle and the glyoxylate cycle, under what circumstances is isocitrate channeled into one pathway versus the other?

Analysis of Experimental Data

36. How the citric acid cycle was discovered.

The detailed mechanism of the citric acid cycle was elucidated through the work of several scientists over the course of several decades. This metabolic pathway was first described by Krebs and Johnson in 1937, based on their own research as well as publications by other investigators.

Research Methods of that era differed markedly from modern biochemical techniques. Prior to the 1940s, radioactive tracers were virtually unused, forcing Krebs and other scientists to employ alternative analytical approaches. Oxygen consumption was measured using freshly prepared pigeon breast muscle preparations; for this, minced Muscle tissue in a buffer was placed in a sealed vessel, and the volume of oxygen (in microliters) consumed under various conditions was determined. For the quantitative analysis of intermediates, the samples were treated with acid to precipitate Proteins, after which the content of low-molecular-weight organic molecules was measured. Two key observations that led Krebs and his colleagues to conclude that The Mechanism of the citric acid cycle is cyclical rather than linear (such as glycolysis) were obtained from the following experiments.

Experiment I. 460 mg of minced muscle tissue was incubated in 3 mL of buffer at 40 °C for 150 min. The addition of citrate increased O₂ consumption by 893 μL compared to control samples without citrate. Based on calculations using the O₂ consumption observed with other carbon sources, the expected O₂ consumption required for the complete oxidation of this amount of citrate was only 302 μL.

Experiment II. Oxygen consumption was determined in the presence of 460 mg of minced muscle tissue in 3 mL of buffer upon incubation with citrate and/or 1-glycerophosphate (known to be readily oxidized during cellular respiration) at 40 °C for 140 min. The experimental results are presented in the table.

a) Why does measuring O₂ consumption provide a reliable estimate of cellular respiration?

b) Why does sample 1 (muscle tissue without added substrates) consume some amount of oxygen?

c) Can it be concluded from the results for samples 2 and 3 that 1-glycerophosphate and citrate act as substrates for cellular respiration in this system? Explain your reasoning.

d) Based on these experimental results, Krebs and his colleagues concluded that citrate possessed "catalytic" properties, meaning it helped the muscle tissue metabolize 1-glycerophosphate more completely. How does this Conclusion follow from the presented data?

e) Krebs and coauthors further argued that citrate is not simply consumed in this process, but must be regenerated. Consequently, the process is cyclical rather than linear. Can you arrive at this conclusion by discussing the provided results?

Other researchers found that arsenate inhibits α-ketoglutarate dehydrogenase activity, while malonate inhibits succinate dehydrogenase activity.

f) Krebs and coworkers discovered that muscle tissue treated with arsenate and citrate consumes citrate only in the presence of oxygen. Based on the data shown in Fig. 16-7, explain what citrate was converted into in this experiment and why the sample consumed oxygen.

Later in their paper, Krebs and Johnson reported the following.

1. In the presence of arsenate, 5.48 mmol of citrate was converted into 5.07 mmol of α-ketoglutarate.

2. In the presence of malonate, citrate was completely converted into a large amount of succinate and a small amount of α-ketoglutarate.

3. The addition of oxaloacetate in the absence of oxygen led to the formation of a large amount of citrate, with the amount of citrate increasing even further upon the addition of glucose to the medium.

For a similar muscle tissue sample, other researchers proposed the following metabolic pathway:

Succinate -> fumarate -> malate -> oxaloacetate -> pyruvate

g) Based solely on the data provided in this problem, indicate the order in which the intermediates of the citric acid cycle are formed. How does this result align with the data presented in Fig. 16-7? Explain your reasoning.

h) Why was it important to demonstrate the quantitative conversion of citrate into α-ketoglutarate?

The paper by Krebs and Johnson also contains much other data that allows

for the complete reconstruction of the cycle pathway. At the time, the only unknown component was the substance that reacts with oxaloacetate to form citrate.