Untitled Notes

Home Explore Exams

Daniel

@danisgreat

view

Rating

0.0(0)

Explore Top Notes

Chapter 1 - The Earth (copy)

Note

Studied by 37 people

4.5 Stars(2)

Unit 3 - Cellular Energetic

Note

Studied by 190 people

Untitled

<html><body>CHAPTER 1 The Citric Acid Cycle 7

The citric acid cycle, also known as the tricarboxylic acid cycleor the Krebs cycle,

is the final oxidative pathway for carbohydrates, lipids, and amino acids. It is also a source of precursors for biosynthesis. The authors begin Chapter 17 with

a detailed discussion of the reaction mechanisms of the pyruvate dehydrogenase complex, followed by a description of the reactions of the citric acid cycle. This description includes details of mechanism and stereospecificity of some of the reactions, and homologies of the enzymes to other proteins. In the following sections, they describe the stoichiometry of the pathway including the energy yield (ATP and GTP) and then describe control mechanisms. They conclude the chapter with a summary of the biosynthetic roles of the citric acid cycle and its relationship to the glyoxylate cycle found in bacteria and plants.

The chapters on enzymes (Chapters 8 through 10), the introduction to metab

olism (Chapter 14), and the chapter on glycolysis (Chapter 16) contain essential background material for this chapter.

When you have mastered this chapter, you should be able to complete the fol

lowing objectives. 287 288

CHAPTER 17 LEARNING OBJECTIVES Introduction

1. Outline the role of the citric acid cycle in aerobic metabolism.

2. Locate the enzymes of the cycle in eukaryotic cells. The Citric Acid Cycle Oxidizes Two-Carbon Units (Text Section 17.1)

3. Account for the origins of acetyl CoA from various metabolic sources.

4. Describe pyruvate dehydrogenase as a multienzyme complex.

5. List the cofactors that participate in the pyruvate dehydrogenase complex reactions and

discuss the roles they play in the overall reaction.

6. Outline the enzymatic mechanism of citrate synthase.

7. Explain the importance of the induced-fit structural rearrangements in citrate synthase

during catalysis.

8. Describe the role of iron in the enzyme aconitase.

9. Compare the reaction catalyzed by the a-ketoglutarate dehydrogenase complex to that cat

alyzed by the pyruvate dehydrogenase complex.

10. Name all the intermediates of the citric acid cycle and draw their structures.

11. List the enzymatic reactions of the citric acid cycle in their appropriate sequence. Name

all the enzymes.

12. Give examples of condensation, dehydration, hydration, decarboxylation, oxidation, and sub- strate-level phosphorylation reactions.

13. Indicate the steps of the cycle that yield CO2, NADH, FADH2, and GTP. Note the bio

logical roles of GTP.

14. Calculate the yield of ATP from the complete oxidation of pyruvate or of acetyl CoA. Entry into the Citric Acid Cycle and Metabolism Through It Are Controlled (Text Section 17.2)

15. Summarize the regulation of the pyruvate dehydrogenase complex through reversible phosphorylation. List the major activators and inhibitors of the kinase and phosphatase.

16. Indicate the control points of the citric acid cycle and note the activators and inhibitors. The Citric Acid Cycle Is a Source of Biosynthetic Precursors (Text Section 17.3)

17. Indicate the citric acid cycle intermediates that may be used as biosynthetic precursors.

18. Describe the role of anaplerotic reactions and discuss the pyruvate carboxylase reaction.

19. Describe the consequences and the biochemical basis of thiamine deficiency. Compare the

effects of heavy metal poisoning with mercury or arsenite.

THE CITRIC ACID CYCLE 289 The Glyoxylate Cycle Enables Plants and Bacteria to Grow on Acetate (Text Section 17.4)

20. Compare the reactions of the glyoxylate cycle and those of the citric acid cycle. List the

reactions that are unique to the glyoxylate cycle. SELF-TEST Introduction

1. If a eukaryotic cell were broken open and the subcellular organelles were separated by

zonal ultracentrifugation on a sucrose gradient, in which of the following would the citric acid cycle enzymes be found?

(a) nucleus

(d) mitochondria

(b) lysosomes

(e) endoplasmic reticulum

2. What are the potential advantages of a multienzyme complex with respect to the isolated

enzyme components? Explain.

3. Match the cofactors of the pyruvate dehydrogenase complex in the left column with their

corresponding enzyme components and with their roles in the enzymatic steps that are listed in the right column.

(a) coenzyme A

(1) pyruvate dehydrogenase component

(b) NAD+

(2) dihydrolipoyl dehydrogenase

(3) dihydrolipoyl transacetylase

(d) FAD

(4) oxidizes the hydroxyethyl group

(e) lipoamide

(5) decarboxylates pyruvate (6) oxidizes dihydrolipoamide (7) accepts the acetyl group from acetyl

lipoamide

(8) provides a long, flexible arm that

conveys intermediates to different enzyme components

(9) oxidizes FADH2

4. Which of the following statements concerning the enzymatic mechanism of citrate syn

thase is correct?

(a) Citrate synthase uses an NAD+ cofactor. (b) Acetyl CoA binds to citrate synthase before oxaloacetate. (c) The histidine residues at the active site of citrate synthase participate in the hy

drolysis of acetyl CoA.

(d) After citryl CoA is formed, additional structural changes occur in the enzyme. (e) Each of the citrate synthase subunits binds one of the substrates and brings the sub

strates into close proximity to each other. 290

CHAPTER 17

5. Citrate synthase binds acetyl CoA, condenses it with oxaloacetate to form citryl CoA,

and then hydrolyzes the thioester bond of this intermediate. Why doesn’t citrate synthase hydrolyze acetyl CoA?

6. Which of the following answers complete the sentence correctly? Succinate dehy

drogenase

(a) is an iron-sulfur protein like aconitase. (b) contains FAD and NAD+ cofactors like pyruvate dehydrogenase. (c) is an integral membrane protein unlike the other enzymes of the citric acid cycle. (d) carries out an oxidative decarboxylation like isocitrate dehydrogenase.

7. The conversion of malate to oxaloacetate has a DGº′ = +7.1 kcal/mol, yet in the citric acid

cycle the reaction proceeds from malate to oxaloacetate. Explain how this is possible.

8. Given the biochemical intermediates of the pyruvate dehydrogenase reaction and the

citric acid cycle (Figure 17.1), answer the following questions: FIGURE 17.1 Citric acid cycle and the pyruvate dehydrogenase reaction.

O

CH JCJCOO:

3

1

O

COO:

CH JCJSJCoA

3

J

CH

2

HJCJCOO:

COO:

α COO:

J

CH

HC

β CKO

2

2

J

K

J

3

COO:

CJCOO:

γ CH

2

J

CH

δ COO:

2

J B

COO:

9

3

COO:

COO:

J

HOJCJH

J

HJCJCOO:

CH

2

J

J

CH2

COO:

J

COO:

8

4

COO:

COO:

J

CKO

CH

7

O

J

COO:

CH

K

2

HC

J

C

J

JSJCoA

5

J

CH

2

J

2

COO:

6

J

CH

J

2

COO:

2

J

CH A

2

COO:

J

COO:

THE CITRIC ACID CYCLE 291

(a) Name the intermediates:

A B

(b) Draw the structure of isocitrate and show those atoms that come from acetyl CoA

in bold letters.

(c) Which reaction is catalyzed by a-ketoglutarate dehydrogenase? (d) Which enzyme catalyzes step 2? (e) Which reactions are oxidations? Name the enzyme catalyzing each of them. (f)

At which reaction does a substrate-level phosphorylation occur? Name the enzyme and the products of this reaction.

(g) Which of the reactions require an FAD cofactor? Name the enzymes. (h) Indicate the decarboxylation reactions and name the enzymes.

9. If the methyl carbon atom of pyruvate is labeled with 14C, which of the carbon atoms of

oxaloacetate would be labeled after one turn of the citric acid cycle? (See the lettering scheme for oxaloacetate in Figure 17.1 in this book.) Note that the “new” acetate carbons are the two shown at the bottom of the first few structures in the cycle, because aconitase reacts stereospecifically.

(a) None. The label will be lost in CO2. (b) a (c) b (d) g (e) d

10. Considering the citric acid cycle steps between a-ketoglutarate and malate, how many

high-energy phosphate bonds, or net ATP molecules, can be generated?

(a) 4

(d) 10

(b) 5

(e) 12

11. The standard free-energy change (in terms of net ATP production) when glucose is con

verted to 6 CO2 and 6 H2O is about how many times as great as the free-energy change when glucose is converted to two lactate molecules?

(a) 2

(b) 7

(d) 28 Entry into the Citric Acid Cycle and Metabolism Through It Are Controlled

12. Although O2 does not participate directly in the reactions of the citric acid cycle, the

cycle operates only under aerobic conditions. Explain this fact.

13. Which of the following answers complete the sentence correctly? The pyruvate dehy

drogenase complex is activated by

(a) phosphorylation of the pyruvate dehydrogenase component (E1). (b) stimulation of a specific phosphatase by Ca2+. (c) inhibition of a specific kinase by pyruvate. (d) decrease of the NADH/NAD+ ratio. (e) decreased levels of insulin. 292

CHAPTER 17

14. First select the enzymes in the left column that regulate the citric acid cycle. Then match

those enzymes with the appropriate control mechanisms in the right column.

(a) citrate synthase

(1) feedback inhibited by succinyl CoA

(b) aconitase

(2) allosterically activated by ADP

(3) inhibited by NADH

(d) a-ketoglutarate dehydrogenase

(4) regulated by the availability of acetyl

(e) succinyl CoA synthetase

CoA and oxaloacetate

(f)

succinate dehydrogenase

(5) inhibited by ATP

(g) fumarase (h) malate dehydrogenase

15. Although the ATP/ADP ratio and the availability of substrates and cycle intermediates are

very important factors affecting the rate of the citric acid cycle, the NADH/NAD+ ratio is of paramount importance. Explain why. The Citric Acid Cycle Is a Source of Biosynthetic Precursors

16. Which of the following statements are correct? The citric acid cycle

(a) does not exist as such in plants and bacteria because its functions are performed by

the glyoxylate cycle.

(b) oxidizes acetyl CoA derived from fatty acid degradation. (c) produces most of the CO2 in anaerobic organisms. (d) provides succinyl CoA for the synthesis of carbohydrates. (e) provides precursors for the synthesis of glutamic and aspartic acids.

17. Match the intermediates of the citric acid cycle in the left column with their biosynthetic

products in mammals, listed in the right column.

(a) isocitrate

(1) aspartic acid

(b) a-ketoglutarate

(2) glutamic acid

(3) cholesterol

(d) cis-aconitate (4) porphyrins (e) oxaloacetate (5) none

18. Which of the following answers complete the sentence correctly? Anaplerotic reactions

(a) are necessary because the biosynthesis of certain amino acids requires citric acid

cycle intermediates as precursors.

(b) can convert acetyl CoA to oxaloacetate in mammals. (c) can convert pyruvate into oxaloacetate in mammals. (d) are not required in mammals, because mammals have an active glyoxylate cycle. (e) include the pyruvate dehydrogenase reaction operating in reverse.

19. Which of the following answers complete the sentence correctly? Pyruvate carboxylase

(a) catalyzes the reversible decarboxylation of oxaloacetate. (b) requires thiamine pyrophosphate as a cofactor. (c) is allosterically activated by NADH. (d) requires ATP. (e) is found in the cytoplasm of eukaryotic cells.

THE CITRIC ACID CYCLE 293

20. Which of the following enzymes have impaired activity in vitamin B1 deficiency?

(a) succinate dehydrogenase (b) pyruvate dehydrogenase (c) isocitrate dehydrogenase (d) a-ketoglutarate dehydrogenase (e) dihydrolipoyl transacetylase (f)

transketolase The Glyoxylate Cycle Enables Plants and Bacteria to Grow on Acetate

21. Malate synthase, an enzyme of the glyoxylate cycle, catalyzes the condensation of gly

oxylate with acetyl CoA. Which enzyme of the citric acid cycle carries out a similar reaction? Would you expect the binding of glyoxylate and acetyl CoA to malate synthase to be sequential? Why?

22. All organisms require three- and four-carbon precursor molecules for biosynthesis, yet

bacteria can grow on acetate whereas mammals cannot. Explain why this is so.

23. Starting with acetyl CoA, what is the approximate yield of high-energy phosphate bonds

(net ATP formed) via the glyoxylate cycle?

(a) 3

(d) 12

(b) 6

(e) 15

1. d

2. A multienzyme complex can carry out the coordinated catalysis of a complex reaction.

The intermediates in the reaction remain bound to the complex and are passed from one enzyme component to the next, which increases the overall reaction rate and minimizes side reactions. In the case of isolated enzymes, the reaction intermediates would have to diffuse randomly between enzymes.

3. (a) 3, 7 (b) 2, 9 (c) 1, 5 (d) 2, 6 (e) 3, 4, 8

4. d

5. Citrate synthase binds acetyl CoA only after oxaloacetate has been bound and the en

zyme structure has rearranged to create a binding site for acetyl CoA. After citryl CoA is formed, there are further structural changes that bring an aspartate residue and a water molecule into the vicinity of the thioester bond for the hydrolysis step. Thus, acetyl CoA is protected from hydrolysis.

6. a, c

7. Although this step is energetically unfavorable at standard conditions, in mitochondria

the concentrations of malate and NAD+ are relatively high and those of the products, oxaloacetate and NADH, are quite low, so the overall DG for this reaction is negative. 294

CHAPTER 17

8. (a) A: a-ketoglutarate; B: oxaloacetate

(b) See the structure of isocitrate in the margin. The text doesn’t go into detail about

the stereochemistry of the enzyme aconitase, but the enzyme always puts the double bond and then the hydroxyl on the side of the molecule away from the “new” carbons introduced from Acetyl CoA.

COO: J

HOJCJH

HJCJCOO:

J CH2

J COO- Isocitrate

(c) reaction 5 (d) citrate synthase (e) step 1, pyruvate dehydrogenase; step 4, isocitrate dehydrogenase; step 5, a-ketoglu

tarate dehydrogenase; step 7, succinate dehydrogenase; step 9, malate dehydrogenase

(f)

step 6; the enzyme is succinyl CoA synthetase; the products of the reaction are succinate, CoA, and GTP.

(g) step 1, dihydrolipoyl dehydrogenase component of the pyruvate dehydrogenase

complex; step 5, dihydrolipoyl dehydrogenase component of the a-ketoglutarate dehydrogenase complex; step 7, succinate dehydrogenase.

(h) step 1, pyruvate dehydrogenase; step 4, isocitrate dehydrogenase; step 5, a-ketog

lutarate dehydrogenase.

9. c and d. Both of the middle carbons of oxaloacetate will be labeled because succinate is

a symmetrical molecule.

10. b

11. c. From glucose to lactate, two ATP are formed; from glucose to CO2 and H2O, about 30

ATP are formed.

12. The citric acid cycle requires the oxidized cofactors NAD+ and FAD for its oxidation

reduction reactions. The oxidized cofactors are regenerated by transfer of electrons through the electron transport chain to O2 to give H2O (see Chapter 18).

13. b, c, d

14. a, c, d. (a) 4, 5 (c) 2, 3, 5 (d) 1, 3, 5

The inhibition of citrate synthase by ATP is species specific (found in certain bacteria), as the text points out (p. 481). Citrate synthase is quite sensitive to the levels of available oxaloacetate and acetyl CoA in all organisms.

15. The oxidized cofactors NAD+ and FAD are absolutely required as electron acceptors in

the various dehydrogenation reactions of the citric acid cycle. When these oxidized cofactors are not available, as when their reoxidation stops in the absence of O2 or respiration, the citric acid cycle also stops.

16. b, e

17. (a) 5 (b) 2 (c) 4 (d) 5 (e) 1

18. a, c

19. a, d

20. b, d, f

21. The condensation of glyoxylate and acetyl CoA carried out by malate synthase in the

glyoxylate cycle is similar to the condensation of oxaloacetate and acetyl CoA carried out by citrate synthase in the citric acid cycle. The initial binding of glyoxylate, which induces structural changes in the enzyme that allow the subsequent binding of acetyl CoA, would be expected in order to prevent the premature hydrolysis of acetyl CoA. See question 5.

22. Bacteria are capable, via the glyoxylate cycle, of synthesizing four-carbon precursor mol

ecules for biosynthesis (e.g., malate) from acetate or acetyl CoA. Mammals do not have an analogous mechanism; in the citric acid cycle, the carbon atoms from acetyl CoA are released as CO2, and there is no net synthesis of four-carbon molecules.

23. a. One NADH is formed that can yield approximately 2.5 molecules of ATP. PROBLEMS

1. In addition to its role in the action of pyruvate dehydrogenase, thiamine pyrophosphate

(TPP) serves as a cofactor for other enzymes, such as pyruvate decarboxylase, which catalyzes the nonoxidative decarboxylation of pyruvate. Propose a mechanism for the reaction catalyzed by pyruvate decarboxylase. What product would you expect? Why, in contrast to pyruvate dehydrogenase, are lipoamide and FAD not needed as cofactors for pyruvate decarboxylase?

2. Sodium fluoroacetate is a controversial poison also known as Compound 1080. When an

isolated rat heart is perfused with sodium fluoroacetate, the rate of glycolysis decreases and hexose monophosphates accumulate. In cardiac cells, fluoroacetate is condensed with oxaloacetate to give fluorocitrate. Under these conditions, cellular citrate concentrations increase, while the levels of other citric acid cycle components decrease. What enzyme is inhibited by fluorocitrate? How can you account for the decrease in glycolysis and the buildup of hexose monophosphates?

3. The conversion of citrate to isocitrate in the citric acid cycle actually occurs by a

dehydration-rehydration reaction with aconitate as an isolatable intermediate. A single enzyme, aconitase, catalyzes the conversion of citrate to aconitate and aconitate to isocitrate. An equilibrium mixture of citrate, aconitate, and isocitrate contains about 90, 4, and 6 percent of the three acids, respectively.

(a) Why must citrate be converted to isocitrate before oxidation takes place in the cit

ric acid cycle?

(b) What are the respective equilibrium constants and standard free-energy changes for

each of the two steps (citrate

aconitate; aconitate

isocitrate)? For the

overall process at 25ºC?

(c) Could the citric acid cycle proceed under standard conditions? Why or why not? (d) Given the thermodynamic data you have gathered about the reactions catalyzed by

aconitase, how can the citric acid cycle proceed under cellular conditions?

4. Lipoic acid and FAD serve as prosthetic groups in the enzyme isocitrate dehydrogenase.

Describe their possible roles in the reaction catalyzed by the enzyme. 296

CHAPTER 17

5. Malonate anion is a potent competitive inhibitor of succinate dehydrogenase, which cat

alyzes the conversion of succinate to malate.

COO:

COO:

J

CH

2

J

COO:

2

J

COO: Succinate Malonate

(a) Why is malonate unreactive? (b) In work that led to the elucidation of the citric acid cycle, Hans Krebs employed

malonate as an inhibitor of succinate dehydrogenase. Earlier studies by Martius and Knoop had shown that in animal tissues there is a pathway from citrate to succinate. Krebs had also noticed that citrate catalytically enhances respiration in minced muscle tissues. Knowing that malonate reduces the rate of respiration in animal cells, he then added citrate to malonate-poisoned muscle. In another experiment, Krebs added fumarate to malonate-poisoned muscle. What changes in succinate concentration did Krebs observe in each of the experiments with malonate-treated muscle, and what was the significance of each finding?

in muscle suspensions if oxaloacetate is added. What is the significance of this experiment, and how did it provide a coherent scheme for terminal oxidation of carbon atoms?

6. Recent studies suggest that succinate dehydrogenase activity is affected by oxaloacetate.

Would you expect the enzyme activity to be enhanced or inhibited by oxaloacetate?

7. Winemakers have to understand some biochemistry to know what is happening as crushed

grapes turn to wine. The major pathway involved is glycolysis, leading to ethanol and CO2 (text pp. 438–439, Section 16.1.9). Early bottling can lead to sparkling wine as more CO2 is produced. A secondary fermentation is allowed to take place in many wines, both red and white, called “malolactic fermentation.” This is classically produced by bacteria that have an enzyme that binds L-malic acid and decarboxylates it to form L-lactate. This process alters the flavor, making the wine more complex and less acidic. The secondary fermentation is so desirable that biotechnologists inserted the gene for this enzyme into Saccharomyces cerevisiae, the yeast used to ferment wine or beer. Initial experiments failed to produce malolactic fermentation using only yeast, but after some thought, researchers inserted another gene into the yeast and the process succeeded.

(a) Why does wine taste less acidic when malate is converted into lactate? (b) What was the second gene that researchers had to insert to make the process work?

8. Oysters and some other molluscs live their adult lives permanently cemented to a

support on the sea floor. The local environment can occasionally become anaerobic. This means that these higher animals have to function as facultative anaerobes (text, p. 427). When oysters are deprived of oxygen, they accumulate succinate. Even though the citric acid cycle cannot be run as a cycle in the absence of oxygen, the reactions can be exploited in a way that maintains redox balance. The “four-carbon” reactions are run backwards, from oxaloacetate to succinate. This produces reduced NAD+ and FAD. Simultaneously, the cycle runs forward from citrate to succinate. This

produces two molecules of NADH. Assuming that the oysters manage a steady supply of oxaloacetate to run these reactions, how much energy would they derive from this process?

9. In the early 1900s, Thunberg proposed a cyclic pathway for the oxidation of acetate. In

his scheme, two molecules of acetate are condensed, with reduction, to form succinate, which in turn is oxidized to yield oxaloacetate. The decarboxylation of oxaloacetate to pyruvate followed by the oxidative decarboxylation of pyruvate to acetate complete the cycle. Assuming that electron carriers like NAD+ and FAD would be part of the scheme, compare the energy liberated by the Thunberg scheme with that liberated by the nowestablished citric acid cycle. Which of the steps in Thunberg’s scheme was not found in subsequent studies?

10. A cell is deficient in pyruvate dehydrogenase phosphate phosphatase. How would such

a deficiency affect cellular metabolism?

11. ATP is an important source of energy for muscle contraction. Pyruvate dehydrogenase

phosphate phosphatase is activated by calcium ion, which increases greatly in concentration during exercise. Why is activation of the phosphatase consistent with the metabolic requirements of muscle during contraction?

12. In addition to the carboxylation of pyruvate, there are other anaplerotic reactions that

help to maintain appropriate levels of oxaloacetate. For example, the respective amino groups of glutamate and aspartate can be removed to yield the corresponding a-keto acids. How can these a-keto acids be used to replenish oxaloacetate levels?

13. The oxidation of a fatty acid with an even number of carbon atoms yields a number of

molecules of acetyl CoA, whereas the oxidation of an odd-numbered fatty acid yields molecules of not only acetyl CoA but also propionyl CoA, which then gives rise to succinyl CoA. Why does only the oxidation of odd-numbered fatty acids lead to the net synthesis of oxaloacetate?

14. Some microorganisms can grow using ethanol as their sole carbon source. Propose a

pathway for the utilization of this two-carbon compound; the pathway should convert ethanol into one or more molecules that can be used for energy generation and as biosynthetic precursors.

15. The citric acid cycle provides most of the energy for eukaryotes. It is how we “make a

living” biochemically. But there are biochemical strategies that are completely unrelated. Bacteria called methanophiles (or methanotrophs) can use methane as a fuel. Propose an energy-conserving reaction sequence for converting methane into CO2. What is the likely yield of ATP of this pathway? ANSWERS TO PROBLEMS

1. The mechanism is similar to that shown on page 469 of the text, in which the C-2 car

banion of TPP attacks the a-keto group of pyruvate. The subsequent decarboxylation of pyruvate is enhanced by the delocalization of electrons in the ring nitrogen of TPP. The initial product is hydroxyethyl-TPP, which is cleaved upon protonation to yield acetaldehyde and TPP. In contrast to the reaction catalyzed by pyruvate dehydrogenase, no net oxidation occurs, so lipoamide and FAD, which serve as electron acceptors, are not needed. 298

CHAPTER 17

2. The accumulation of citrate and the decrease in the levels of other citric acid cycle in

termediates suggest that aconitase is inhibited by fluorocitrate. Excess citrate inhibits phosphofructokinase, causing a decrease in the rate of glycolysis and an accumulation of hexose monophosphates such as glucose 6-phosphate and fructose 1,6-bisphosphate. The controversy over Compound 1080 is between environmentalists, who want it banned from outdoor use, and farmers and ranchers, who find it useful against rodents and predators. Because of the way it acts on cells, there is no antidote and it produces a slow and painful death.

3. (a) The oxidation of isocitrate involves oxidation of a secondary alcohol. Citrate has

an alcohol function, but it is a tertiary alcohol, which is much more difficult to oxidize. Isomerization of citrate to isocitrate provides an easier route to oxidative decarboxylation.

(b) For the citrate-aconitate pair, the equilibrium constant is equal to the ratio of prod

uct and substrate concentration.

aconitate

[

]

4 K

=

= .

4 44 ×

−2

eq

10

citrate

[

]

90

The value of DGº′ is −1.36 log10 (4.44 × 10−2) = −1.36(−1.35) = +1.84 kcal/mol.

Similar calculations for the aconitate-isocitrate pair give K

=

eq

6/4 = 1.5 and of

DGº′ = −0.24 kcal/mol.

The overall standard free-energy value for the conversion of citrate to isocitrate is the sum of the two values for the individual reactions.

of DGº′ = 1.84 + (−0.24) = +1.60 kcal/mol

itive free-energy value for the reaction indicates that it would proceed toward net formation of citrate. Note that under standard conditions, everything would be present at 1 molar concentration.

(d) The net conversion of citrate to isocitrate can occur in the mitochondrion if the isoc

itrate produced is then converted to a-ketoglutarate. This would lower the concentration of isocitrate, pulling the reaction toward net formation of that molecule. Concentrations of citrate could also be increased, driving the reaction once again toward the formation of isocitrate. Although accurate concentrations of metabolites in mitochondria are difficult to establish, it appears that both mechanisms may operate to ensure net isocitrate synthesis.

4. Lipoic acid contains a sulfhydryl group that could act as an acceptor for electrons from

isocitrate. Those electrons could then be transferred to NAD+ via FAD. The roles of the prosthetic groups would be similar to those they play in the reaction catalyzed by pyruvate dehydrogenase.

5. (a) Succinate, which has two methylene groups, loses two hydrogens during its oxida

tion by succinate dehydrogenase. Malonate, which has only one methylene group, cannot be dehydrogenated and is therefore unreactive.

THE CITRIC ACID CYCLE 299

(b) In both experiments, Krebs observed an increase in the concentration of succinate.

We know now that both citrate and fumarate can be viewed as precursors of succinate or other components of the citric acid cycle. A malonate-induced block in the conversion of succinate to fumarate would cause an increase in succinate concentration. The first experiment, in which citrate addition caused an increase in succinate concentration, showed that the pathway from citrate to succinate is physiologically significant and is related to the process of respiration using carbohydrates as a fuel. The second experiment with fumarate suggested that a pathway from fumarate to succinate exists which is separate from the reaction catalyzed by succinate dehydrogenase. Krebs realized that a cyclic pathway could account for all these observations. The piece of the puzzle that was left was to learn how citrate might be generated from pyruvate or acetate. The results from those experiments are described in (c).

incorporated two-carbon molecules from acetate or pyruvate into citrate, with oxaloacetate serving as an acceptor of those carbon atoms. He then was able to use the results of his experiments and those of others to show how a cyclic pathway could function to carry out oxidation of carbon molecules while regenerating oxaloacetate. Krebs was prepared for the development of the cyclic scheme because he had shown earlier that the ornithine cycle, which is used for urea synthesis, is also a cyclic metabolic pathway. Krebs’s famous paper, which describes the entire cycle as well as the malonate inhibition study, appeared in Enzymologia 4(1937):148.

6. Oxaloacetate is derived from succinate by the sequential action of succinate dehydroge

nase, fumarase, and malate dehydrogenase. When levels of oxaloacetate are high, one would expect the activity of the enzyme to be reduced. Low levels of oxaloacetate would call for an increase in succinate production.

7. (a) Malic acid has two carboxyl groups. Lactic acid has only one. The pH of the wine

changes significantly as malate is converted into lactate and carbon dioxide.

(b) The yeast cells with the extra gene for the malolactic enzyme were quite capable of

running the reaction, but they had no transport system for malate. The researchers realized that they could also insert a gene for malate permease, which would allow malate to enter the yeast cells and be metabolized. With both genes, the system started working well. An example of a malate permease is shown in Figure 18.38, page 515 (Nature Biotech. 15[1997]:224, 253).

8. One high-energy bond is generated by succinyl CoA synthetase, which produces GTP.

And notice that while the NADH used up cancels out the NADH produced, we have a second NADH that can provide electrons through the electron transport chain to reduce FAD (see Chapter 18, p. 498). The passage of a pair of electrons forward through Complex I (NADH-Q reductase) and backward through Complex II (Succinate-Q reductase) should produce enough of a proton gradient to form another ATP. The details of ATP production in mitochondria will be discussed in Chapter 18, but the basic facts are described in Section 17.1.9 of the text. Metabolism in facultative anaerobes is discussed in J. Biol. Chem. 251(1976):3599. 300

CHAPTER 17

9. As shown in Figure 17.2, there are at least four steps that generate reduced electron

carriers. For each acetate group consumed, 3 NADH and 1 FADH2 are generated, and their subsequent reoxidation in the electron transport chain provides energy for the generation of nine molecules of ATP. The same number of reduced electron carriers is generated through the action of pyruvate dehydrogenase and the enzymes of the citric acid cycle, so that the energy liberated by both schemes is the same. Each of the reactions shown in Thunberg’s scheme is known to occur, except for the condensation of two acetyl groups to form succinate. FIGURE 17.2 Thunberg’s cycle.

CJSJCoA

J

CO

2

CKO Acetyl CoA

NADH

J

CH3

CH

3

COO:

J

CoA

COO:

J

CKO Acetate

CO

2

J

CH3

NADH Pyruvate

COO:

COO:

J

CKO

CH2

J

CH

2

J

COO:

COO: Oxaloacetate Succinate

COO:

NADH

J

CH

COO:

K

FADH

2

J

HC

HOJCJH

J

COO:

CH2

H O

2

J Fumarate

COO: Malate

10. Pyruvate dehydrogenase phosphate phosphatase removes a phosphoryl group from pyru

vate dehydrogenase, activating the enzyme complex and accelerating the rate of synthesis of acetyl CoA. Cells deficient in phosphatase activity cannot activate pyruvate dehydrogenase, so that the rate of entry of acetyl groups into the citric acid cycle will decrease, as will aerobic production of ATP. Under such conditions, stimulation of glycolytic activity and a subsequent increase in lactate production would be expected as the cell responds to a continued requirement for ATP synthesis. See the clinical note on page 481 of the text (Section 17.2.1).

11. As discussed in the previous problem, the phosphatase activates pyruvate dehydroge

nase, stimulating the rate of both glycolysis and the citric acid cycle. Calcium-mediated activation of pyruvate dehydrogenase therefore promotes increased production of ATP, which is then available for muscle contraction.

12. Examination of the structures of the a-keto acid analogs of glutamate and aspartate shows

that they are in fact both citric acid cycle intermediates, a-ketoglutarate and oxaloacetate.

THE CITRIC ACID CYCLE 301

Aspartate, when it is deaminated, thus contributes directly to the insertion of additional molecules of oxaloacetate. Glutamate produces a-ketoglutarate, which, as a component of the citric acid cycle, is a precursor of oxaloacetate.

NH ;

O

3

J

K

J

HJCJCOO:DNH ;+ CJCOO:

HJCJCOO:DNH ;+ CJCOO:

4

J

CH

2

J

CH

COO:

COO:

2

J

J

COO:

COO: Glutamate a-Ketoglutarate Aspartate Oxaloacetate

13. The entry of acetyl groups from acetyl CoA into the citric acid cycle does not contribute

to the net synthesis of oxaloacetate, because two carbons are lost as CO2 in the pathway from citrate to oxaloacetate. Only the entry of compounds with three or more carbons, like succinate, can increase the relative number of carbon atoms in the pathway. Thus, while odd-numbered fatty acids contribute to the net synthesis of oxaloacetate, those compounds with an even number of fatty acids do not.

14. The microorganism first converts ethanol to acetic acid, or acetate, by carrying out two

successive oxidations, with acetaldehyde as an intermediate. Two molecules of a reduced electron carrier such as NADH will also be produced. Next, acetate is activated through the action of acetyl CoA synthetase to form acetyl CoA, and then the acetyl group is transferred to oxaloacetate to form citrate. After citrate is converted to isocitrate, two enzymes from the glyoxylate cycle, isocitrate lyase and malate synthase, assist in the net formation of oxaloacetate from isocitrate and another molecule of acetyl CoA, as discussed in the text on page 485. Oxaloacetate can then be used for generation of energy as well as production of biosynthetic intermediates. Note that a small amount of oxaloacetate and other intermediates of the citric acid cycle must be present initially in order for acetate to enter the pathway.

15. Methane is first oxidized by a monooxygenase to methanol; NADH, the reductant, is

converted to NAD+ while methanol and water are generated. Methanol is then oxidized to formaldehyde; PQQ, a novel quinone, is the electron acceptor in this step. Formaldehyde is oxidized to formic acid, which in turn is oxidized to CO2. NADH is formed in each of these two steps. About four ATP are formed (2.5 ATP from NADH and 1.5 from PQQH2). See G. Gottschalk (1986), Bacterial Metabolism (2nd ed., pp. 155, 163), Springer-Verlag. EXPANDED SOLUTIONS TO TEXT PROBLEMS

1. To answer this problem one must follow carbon atoms around the citric acid cycle as

shown by Figure 17.15 of your text. Remember that the randomization of carbon occurs at succinate, a truly symmetrical molecule. Also, this problem (and the answers given) assumes that all pyruvate goes to acetyl CoA. In fact, this is not necessarily true, since pyruvate can also enter the cycle at oxaloacetate.

(a) After one round of the citric acid cycle, the label emerges in C-2 and C-3 of

oxaloacetate.

(b) After one round of the citric acid cycle, the label emerges in C-1 and C-4 of

oxaloacetate. 302

CHAPTER 17

(c) The label emerges in CO2 in the formation of acetyl CoA from pyruvate. (d) The fate is the same as in (a). (e) C-1 of G-6-P becomes the methyl carbon of pyruvate and hence has the same fate

as in (a).

2. (a) Isocitrate lyase and malate synthase are required in addition to the enzymes of the

citric acid cycle.

(b) 2 acetyl CoA + 2 NAD+ + FAD + 3 H2O

oxaloacetate + 2 CoA + 2 NADH + FADH +

2

3H+

oxylate cycle.

3. Addition of the DGº′ values in Table 17.2 of the text gives the answer −9.8 kcal/mol. 4. As with enzymes, the small-molecule intermediates in the citric acid cycle are not

consumed. Rather the cycle intermediates “turn over,” that is, each of them is regenerated at a particular point during each turn of the cycle. The cycle as a whole catalyzes the conversion of acetyl-coenzyme A into two molecules of CO2, with the release of free coenzyme A and the concomitant production of GTP, FADH2, and three molecules of NADH.

5. The coenzyme stereospecificity of glyceraldehyde 3-phosphate dehydrogenase is the op

posite of that of alcohol dehydrogenase (type B versus type A, respectively).

6. Thiamine thiazolone pyrophosphate is a transition state analog. The sulfur-containing

ring of this analog is uncharged, and so it closely resembles the transition state of the normal coenzyme in thiamine-catalyzed reactions. See J. A. Gutowski and G. E. Lienhard, J. Biol. Chem. 251(1976):2863, for a discussion of this analog.

7. Without O2 as a terminal acceptor for electrons from NADH, the citric acid cycle can

not operate in a sustained manner. Rather, the pyruvate that is produced by glycolysis must be reduced to lactate (in muscle; or ethanol in yeast) so that the NADH produced in glycolysis can be oxidized to NAD+. Oxygen deficiency is made worse by the presence of carbon dioxide, which, along with acetyl-CoA, is a product of the pyruvate dehydrogenase complex. Therefore, inhibiting pyruvate dehydrogenase will decrease the production of CO2 and lessen the severity of the shock.

8. (a) The steady-state concentrations of the products (OAA + NADH) are much lower

than those of the substrates (Mal + NAD+). Hence, the reaction is pushed “uphill” by the overwhelming mass action caused by the differences in concentration.

(b)

[OAA][

]

NADH

7 / − .

= 10 1 36 = .

−

7 08 ×

6

10

[

]

Mal [NAD+ ]

Since

+

[NADH]/ [NAD ] = 1 / ,

8

[OAA] = .

−

7 08 ×

6

10

× =

.

−

5 67 ×

5

10

[

]

Mal

The reciprocal of this is 1.75 × 104, the smallest [Mal]/[OAA] ratio permitting net OAA formation.

THE CITRIC ACID CYCLE 303

9. We need a scheme for the net synthesis of a-ketoglutarate from pyruvate, that is, we seek

reactions that will allow all of the carbons in a-ketoglutarate to come from pyruvate. This will be possible only if half of the available pyruvate is converted to oxaloacetate by the anaplerotic reaction (pyruvate carboxylase), while the other half is converted to acetyl-CoA by pyruvate dehydrogenase. Here is the set of reactions that must be summed:

Pyruvate + CO +

+

2

ATP + H2O

oxaloacetate + ADP + Pi

2H+

Pyruvate + CoA + NAD+

acetyl-CoA + CO +

2

NADH

Acetyl-CoA + oxaloacetate + H2O

citrate + CoA + H+

Citrate

isocitrate

Isocitrate + NAD+

a-ketoglutarate + CO +

2

NADH

Sum: 2 Pyruvate + ATP + 2 NAD+ + 2 H2O

a-ketoglutarate + CO +

+

2

ADP + Pi

2 NADH + 3H+

10. We cannot get the net conversion of fats into glucose, because the only means to get the

carbons from fats into oxaloacetate, the precursor to glucose, is through the citric acid cycle. However, although two carbon atoms enter the cycle as acetyl CoA, two carbon atoms are lost as CO2 before the oxaloacetate is formed. Thus, although some carbon atoms from fats may end up as carbon atoms in glucose, we cannot obtain a net synthesis of glucose from fats.

11. The enolate anion of acetyl CoA attacks the carbonyl carbon atom of glyoxylate to form

a C−C bond. This reaction is like the condensation of oxaloacetate with the enolate anion of acetyl CoA. Glyoxylate contains a hydrogen atom in place of the −CH2COO− of oxaloacetate; the reactions are otherwise nearly identical.

12. The labeled carbon will be incorporated into citrate at carbon 5 (only):

1

COO:

2

:

3

OOCJJJJ OH

6

4

COO:

*citrate labeled at carbon 5

5*

In the drawing, carbons 1 and 2 of citrate come from acetyl-CoA. Carbon 6 is lost in the formation of a-ketoglutarate, so none of the label from carbon 5 is lost in that step. Early investigators (until Ogston in 1948; see next problem) thought that carbons 1 and 5 of citrate were indistinguishable and so were surprised when all of carbon 5 and none of carbon 1 was lost in the decarboxylation of a-ketoglutarate. In fact, citrate is prochiral and so the two ends are distinguishable (see next problem). 304

CHAPTER 17

13. The enzyme can provide a “3-point landing” at sites X′, Y′, and H′, to bind groups X, Y,

and always the lower H (never the upper H on the small molecule). The two hydrogens on the tetrahedral carbon atom in the drawing are therefore distinguishable based on their relative orientations with respect to X and Y. The molecule CXYH2 is “prochiral.”

H

J

X

Y

H

X„

Y„

H„

14. (a) A balanced equation for the oxidation of citrate would be

C

+

6H8O7

4.5 O2

4 H2O + 6 CO2

From the stoichiometry of the balanced equation, 4.5 moles of O2 would be consumed per mole of citrate, corresponding to 13.5 mmol O2 per 3 mmol citrate.

(b) The consumption of oxygen is higher than a stoichiometric oxidation of citrate

would suggest. The result could suggest that the citrate is not being consumed, but rather is acting “catalytically” or is being regenerated in a cycle (as Krebs correctly hypothesized).

15. (a) The presence of arsenite correlates with the disappearance of citrate.

(b) When more citrate is present, a smaller fraction of the total citrate disappears (38%

of 90 mmol disappears, whereas 95% of 22 mmol disappears).

cycle is inhibited by arsenite. (If the immediate step citrate

isocitrate step were

inhibited, then citrate would accumulate, but this is not observed.) At low citrate concentrations, the citrate disappears almost completely because its regeneration is blocked (as some “downstream” step of the cycle is blocked by arsenite). At higher citrate concentrations, some steps between citrate and the block may reversibly approach equilibrium; in this case not all citrate would be depleted.

16. (a) The number of bacterial colony-forming units is much lower in the absence of the

gene for isocitrate lyase. (After 15 weeks, the difference is a factor of 100, i.e., fewer than 105 cfu without the gene, compared to >107 cfu when the gene is present.)

(b) Yes. When the isocitrate lyase gene is restored, then the number of cfu also is restored. (c) The experiment in part b confirms the direct influence of the gene for isocitrate

lyase. (Because replacing the gene restores the number of CFU, other possible indirect factors or unexpected side effects of removing the gene can be excluded.)

(d) The glyoxalate cycle will allow the bacteria to subsist on acetate from the break

down of fatty acids from the lipid-rich environment. Without the glyoxalate cycle, the synthesis of carbohydrates from lipids is not possible. One can speculate that without the glyoxalate cycle, the bacteria will lack carbohydrates or other key metabolic intermediates. </body></html>

Home