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Chapter 10. The Citric Acid Cycle. The Citric Acid Cycle. The common pathway leading to complete oxidation of carbohydrates, fatty acids, and amino acids to CO 2 . A pathway providing many precursors for biosynthesis.

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Chapter 10

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Chapter 10

Chapter 10

The Citric Acid Cycle

The Citric Acid Cycle

The common pathway leading to complete oxidation of carbohydrates, fatty acids, and amino acids to CO2.

A pathway providing many precursors for biosynthesis.


Chapter 10

1.The cellular respiration (complete oxidation of fuels) can be divided into three stages

Stage I All the fuel molecules are oxidized to generate a common two-carbon unit, acetyl-CoA.

Stage II The acetyl-CoA is completely oxidized into CO2, with electrons collected by NAD and FAD via a cyclic pathway (named as the citric acid cycle, Krebs cycle, or tricarboxylic acid cycle).

Stage III Electrons of NADH and FADH2 are transferred to O2 via a series carriers, producing H2O and a H+ gradient, which will promote ATP formation.

The Citric Acid Cycle


Chapter 10

1.The cellular respiration (complete oxidation of fuels) can be divided into three stages

The Citric Acid Cycle

Mitochondria is the major site for

fuel oxidation to generate ATP.


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

Pyruvate is first transported into mitochondria via a specific transporter on the inner membrane.

Pyruvate is converted to acetyl-CoA and CO2 by oxidative decarboxylation.

The pyruvate dehydrogenasecomplex is a huge multimeric assembly of three kinds of enzymes, having 60 subunits in bacteria and more in mammals.

Pyruvate is first decarboxylated after binding to the prosthetic group (辅基,TPP) of pyruvate dehydrogenase (E1), forming hydroxyethyl-TPP.(羟乙基-焦磷酸硫胺素)

The Citric Acid Cycle


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The hydroxyethyl group attached to TPP is oxidized and transferred: First two electrons, then the acetyl group formed are all transferred to the lipoyllysyl (硫辛酰赖氨酰)group of dihydrolipoyl transacetylase (二硫辛酰转乙酰基酶,E2).

The lipoyllysyl group serves as both electron and acetyl carriers.

The acetyl group is then transferred (still catalyzed by E2) from acetyllipoamide乙酰硫辛酰胺to CoA-SH, forming acetyl-CoA.

The oxidized lipoamide group is then regenerated by the action of dihydrolipoyl dehydrogenase (二硫辛酰脱氢酶,E3), with electrons collected by FAD and then by NAD+.

The Citric Acid Cycle


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

Substrates of the five reactions catalyzed by pyruvate dehydrogenase complex are efficiently channeled : The lipoamide group attached to E2 swings between E1 (accepting the electrons and acetyl group) and E3 (giving away the electrons), passing the acetyl group to Coenzyme A on E2

The multienzyme complexes catalyzing the oxidative decarboxylation of a few different kinds of α-keto acids, pyruvate dehydrogenase complex, α -ketoglutarate(酮戊二酸) dehydrogenase complex and branched chain a-keto acid dehydrogenase complex show remarkable structure and function relatedness (all have identical E3, similar E1 and E2).

The Citric Acid Cycle


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The Citric Acid Cycle

The oxidative decarboxylation of pyruvate

in mitochondria: producing acetyl-CoA and CO2.


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The Citric Acid Cycle


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The Citric Acid Cycle

Electron micrograph of pyruvate

dehydrogenase complexes from E. coli


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The Citric Acid Cycle


Chapter 10

pyruvate

acetyl-CoA

CO2

hydroxyethyl-TPP

E3

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

E2 (dihydrolipoyl transacetylase):

consisting the core, 24 subunits;

E1 (pyruvate dehydrogenase):

bound to the E2 core, 24 subunits;

E3 (dihydrolipoyl dehydrogenase):

bound to the E2 core, 12 subunits.

(a protein kinase and

phosphoprotein phosphatase, not

shown here, are also part of the

complex)

The Citric Acid Cycle

A model of the E. coli pyruvate dehydrognase complexshowing the three kinds of enzymes and the flexible lipoamide arms covalently attached to E2.


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The E2 core (a total of 24 subunits) forms a hollow cube.

The Citric Acid Cycle

X-ray structure of the E2 transacetylase core: Only four out of eight trimers are shown here.


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The Citric Acid Cycle

The oxidative decarboxylation of pyruvate is catalyzed by a multiezyme complex: pyruvate dehydrogenase complex.


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The Citric Acid Cycle


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The Citric Acid Cycle

With the help of TPP, pyruvate is decarboxylated: identical reaction as catalyzed by pyruvate decarboxylase.


Chapter 10

Dihydrolipoyl

The lipoyllysyl group

serves as the electron

and acetyl carriers

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The Citric Acid Cycle


Chapter 10

2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex

The Citric Acid Cycle


Chapter 10

3. The complete oxidation of pyruvate in animal tissues was proposed to undergo via a cyclic pathway

O2 consumption and pyruvate oxidation in minced muscle tissues

were found to be stimulated by some four-carbon dicarboxylic

acids (Fumarate, succinate, malate and oxaloacetate, five-

carbon dicarboxylic acid (a-ketoglutarate ), or six-carbon

tricarboxylic acids (citrate, isocitrate, cis-aconitate).

A small amount of any of these organic acids stimulates many

folds of pyruvate oxidation!

Malonate(丙二酸) inhibits pyruvate oxidation regardless of

which active organic acid is added!

The Citric Acid Cycle


Chapter 10

3. The complete oxidation of pyruvate in animal tissues was proposed to undergo via a cyclic pathway

Hans Krebs proposed the “citric acid cycle” for the complete

oxidation of pyruvate in animal tissues in 1937 (he wrongly

hypothesized that pyruvate condenses with oxaloacetate in his

original proposal).

The citric acid cycle was confirmed to be universal in cells by

in vitro studies with purified enzymes and in vivo studies with

radio isotopes (“radio isotope tracer experiments”).

Krebs was awarded the Nobel prize in medicine in 1953 for

revealing the citric acid cycle (thus also called the Krebs

cycle).

The Citric Acid Cycle


Chapter 10

3. The complete oxidation of pyruvate in animal tissues was proposed to undergo via a cyclic pathway

The Citric Acid Cycle


Chapter 10

4. The acetyl group (carried by CoA) is completely oxidized to CO2via the citric acid cycle

  • The 4-carbon oxaloacetate (草酰乙酸) acts as the “carrier” for the oxidation.

  • The two carbons released as 2 CO2 in the first cycle of oxidation are not from the acetyl-CoA just joined.

  • The 8 electrons released are collected by three NAD+ and one FAD.

  • One molecule of ATP (or GTP) is produced per cycle by substrate-level phosphorylation.

The Citric Acid Cycle


Chapter 10

4. The acetyl group (carried by CoA) is completely oxidized to CO2via the citric acid cycle

The Citric Acid Cycle


Chapter 10

4. The acetyl group (carried by CoA) is completely oxidized to CO2via the citric acid cycle

The Citric Acid Cycle


Chapter 10

5. The citric acid cycle consists of eight successive reactions

Step 1 The methyl carbon of acety-CoA joins the carbonyl carbon of oxaloacetate via aldol condensation to form citrate (柠檬酸); citroyl-CoA is a transiently intermediate but hydrolyzed immediately in the active site of citrate synthase; hydrolysis of the thioester bond releases a large amount of free energy, driving the reaction forward; large conformational changes occur after oxaloacetate is bound and after citroyl-CoA is formed, preventing the undesirable hydrolysis of acetyl-CoA.

The Citric Acid Cycle


Chapter 10

5. The citric acid cycle consists of eight successive reactions

Step 2 Citrate is isomerized into isocitrate (get the six-carbon unit ready for oxidative decarboxylation) via a dehydration step followed by a hydration step; cis-aconitate (顺乌头酸) is an intermediate during this transformation, thus the catalytic enzyme is named as aconitase, which contains a 4Fe-4S iron-sulfur center directly participating substrate binding and catalysis.

Step 3 Isocitrate is first oxidized and then decarboxylated to form α-ketoglutarate (a-酮戊二酸); oxalosuccinate is an intermediate; two electrons are collected by NAD+; the carbon released as CO2 is not from the acetyl group joined; catalyzed by isocitrate dehydrogenase.

The Citric Acid Cycle


Chapter 10

5. The citric acid cycle consists of eight successive reactions

Step 4α-ketoglutarate undergoes another round of oxidative decarboxylation; decarboxylated first, then oxidized to form succinyl-CoA (琥珀酰辅酶A); again the carbon released as CO2 is not from the acetyl group joined; catalyzed by a-ketoglutarate dehydrogenase complex; reactions and enzymes closely resemble pyruvate dehydrogenase complex (with similar E1 and E2, identical E3).

Step 5 Succinyl-CoA is hydrolyzed to succinate (琥珀酸或戊二酸); the free energy released by hydrolyzing the thioesterbond is harvested by a GDP or an ADP to form a GTP or an ATP by substrate-level phosphorylation; the reversible reaction is catalyzed by succinyl-CoA synthetase (or succinic thiokinase ,琥珀酸硫激酶);

The Citric Acid Cycle


Chapter 10

5. The citric acid cycle consists of eight successive reactions

acyl phosphate and phophohistidyl enzyme are intermediates; the active site is located at the interface of two subunits; the negative charge of the phospho-His intermediate is stabilized by the electric dipoles of two a helices (one from each subunit).

Step 6 Succinate is oxidized to fumarate (延胡索酸或反丁烯二酸); catalyzed by a flavoprotein succinate dehydrogenase (with a covalently bound FAD and three iron-sulfur centers), which is tightly bound to the inner membrane of mitochondria; malonate (丙二酸) is a strong competitive inhibitor of the enzyme, that will block the whole cycle.

The Citric Acid Cycle


Chapter 10

5. The citric acid cycle consists of eight successive reactions

Step 7 Fumarate (延胡索酸) is hydrated to L-malate(苹果酸)by the action of fumarase (延胡索酸酶); the enzyme is highly stereospecific, only act on the trans and L isomers, not on the cis and D isomers (maleate and D-malate);

Step 8Oxaloacetate is regenerated by the oxidation of L-malate; this reaction is catalyzed by malate dehydrogenase with two electrons collected by NAD+.

The Citric Acid Cycle


Chapter 10

5. The citric acid cycle consists of eight successive reactions

The aldol condensation between acetyl-CoA and

oxaloacetate forms citrate

The Citric Acid Cycle


Chapter 10

5. The citric acid cycle consists of eight successive reactions

Citrate synthase before

and after binding to

oxaloacetate

The Citric Acid Cycle

Oxaloacetate

Carboxylmethyl-CoA


Chapter 10

5. The citric acid cycle consists of eight successive reactions

Citrate is converted to isocitrate via dehydration followed by a

Hydration(水合作用) step.

The Citric Acid Cycle

4Fe-4S cubic array:each Fe is bonded to three inorganic S and a cysteine sulfur atom (except one)


Chapter 10

5. The citric acid cycle consists of eight successive reactions

The Citric Acid Cycle

The first

oxidation step

Isocitrate is converted to a-ketoglutarate via an

oxidative decarboxylation step, generating NADH

CO2.


Chapter 10

5. The citric acid cycle consists of eight successive reactions

TPP lipoate, FAD

The Citric Acid Cycle

(E1, E2, E3)

The second

oxidation step

Thea-ketoglutarate dehydrogenase complex

closely resembles the pyruvate dehyrogenase

complex in structure and function


Chapter 10

5. The citric acid cycle consists of eight successive reactions

Succinyl-CoA synthetase

catalyzes the substrate-level

phosphorylation of ADP.

The Citric Acid Cycle


Chapter 10

5. The citric acid cycle consists of eight successive reactions

Succinyl-CoA Synthetase from E. coli

The Citric Acid Cycle

Coenzyme A

His246-Pi

The power helices


Chapter 10

5. The citric acid cycle consists of eight successive reactions

The Citric Acid Cycle

The third oxidation step

(An enzyme bound to

the inner membrane

of mitochondria)


Chapter 10

5. The citric acid cycle consists of eight successive reactions

The Citric Acid Cycle

(a stereospecific enzyme)


Chapter 10

5. The citric acid cycle consists of eight successive reactions

The Citric Acid Cycle

(The fourth oxidation

Step in the cycle)

Oxaloacetate is regenerated at the end


Chapter 10

6. The complete oxidation of one glucose may yield as many as 32 ATP

  • All the NADH and FADH2 will eventually pass their electrons to O2 after being transferred through a series of electron carriers.

  • The complete oxidation of each NADH molecule leads to the generation of about 2.5 ATP, and FADH2 of about 1.5 ATP.

  • Overall efficiency of energy conservation is about 34% using the free energy changes under standard conditions and about 65% using actual free energy changes in cells.

The Citric Acid Cycle


Chapter 10

6. The complete oxidation of one glucose may yield as many as 32 ATP

The Citric Acid Cycle


Chapter 10

7. The citric acid intermediates are important sources for biosynthetic precursors

  • The citric acid cycle is the hub of intermediary metabolism serving both the catabolic分解代谢的and anabolic合成代谢的processes .

  • It provides precursors for the biosynthesis of glucose, amino acids, nucleotides, glucose, fatty acids, sterols, heme groups, etc.

  • Intermediates of the citric acid cycle get replenished (充满的)by anaplerotic(补缺的)reactions when consumed by biosynthesis.

The Citric Acid Cycle


Chapter 10

8. The pyruvate dehydrogenase complex in vertebrates is regulated alloseterically and covalently

  • The formation of acetyl-CoA from pyruvate is a key irreversible step in animals because they are unable to convert acetyl-CoA into glucose.

  • The complex (in all organisms) is allosterically inhibited by signaling molecules indicating a rich source of energy, e.g., ATP, acetyl-CoA, NADH, fatty acids; activated by molecules indicating a lack (or demand) of energy, e.g., AMP, CoA, NAD+, Ca2+.

The Citric Acid Cycle


Chapter 10

8. The pyruvate dehydrogenase complex in vertebrates is regulated alloseterically and covalently

  • The activity of the complex (in vertebrates, probably also in plants, but not in E. coli) is also regulated by reversible phosphorylation of one of the enzymes, E1, in the complex: phosphorylation of a specific Ser residue inhibits and dephosphorylation activates the complex.

  • The kinase and phosphatase is also part of the enzyme complex.

  • The kinase is activated by a high concentration of ATP.

The Citric Acid Cycle


Chapter 10

8. The pyruvate dehydrogenase complex in vertebrates is regulated alloseterically and covalently

The Citric Acid Cycle


Chapter 10

9. The rate of the citric acid cycle is controlled at three exergonic irreversible steps

  • Citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase;

  • Inhibited by product feedback (citrate, succinyl-CoA) and high energy charge (ATP, NADH);

  • Activated by a low energy charge (ADP) or a signal for energy requirement (Ca2+).

The Citric Acid Cycle


Chapter 10

10. The partitioning of isocitrate, between the citric acid and glyoxylate乙醛酸cycles is coordinately regulated

  • The activity of the E. coli isocitrate dehydrogenase is inhibited when phosphorylated by a specific kinase and activated when dephosphorylated by a specific phosphatase.

  • The kinase and phosphatase activities are located in two domains of the same polypeptide and are reciprocally regulated: the kinase is allosterically inhibited (while the phosphatase activated) by molecules indicating an energy depletion, e.g., accumulation of intermediates of glycolysis and citric acid cycle.

  • The allosteric inhibitors of the kinase also act as inhibitors for the lyase: i.e., they activate the dehydrogenase while simultaneously inhibit the lyase.

The Citric Acid Cycle


Chapter 10

10. The partitioning of isocitrate, between the citric acid and glyoxylate乙醛酸cycles is coordinately regulated

The Citric Acid Cycle

The isocitrate dehydrogenase and

the isocitrate lyase are coordinately

regulated.


Chapter 10

Summary

  • Pyruvate is converted to acetyl-CoA by the action of pyruvate dehydrogenase complex, a huge enzyme complex.

  • Acetyl-CoA is converted to 2 CO2 via the eight-step citric acid cycle, generating three NADH, one FADH2, and one ATP (by substrate-level phophorylation).

  • Intermediates of citric acid cycle are drawn off to synthesize many other biomolecules, including fatty acids, steroids, amino acids, heme, pyrimidines, and glucose.

The Citric Acid Cycle


Chapter 10

Summary

  • Oxaloacetate can get supplemented from pyruvate, via a carboxylation reaction catalyzed by the biotin-containing pyruvate carboxylase羧化酶.

  • The activity of pyruvate dehydrogenase complex is regulated by allosteric effectors and reversible phosphorylations.

  • Net conversion of fatty acids to glucose can occur in germinating seeds, some invertebrates and some bacteria via the glycoxylate cycle, which shares three steps with the citric acid cycle but bypasses the two decarboxylation steps, converting two molecules of acetyl-CoA to one succinate.

  • Acetyl-CoA is partitioned into the glyoxylate乙醛酸cycle and citric acid cycle via a coordinately regulation of the isocitrate dehydrogenase and isocitrate lyase.

The Citric Acid Cycle


Chapter 10

References

  • De Kok, A., Hangeveld, A. F., Martin, A., and Westphal, A. H. (1998) “The pyruvate dehydrogenase multienzyme complex from gram-negative bacteria” Biochim. Biphys. Acta 1385:353-366.

  • Hagerhall, C. (1997) “Succinate:quinone oxidoreductase. Variations on a conserved theme” Biochim. Biophys. Acta 1320:107-141.

  • Knowles, J. (1989) “The mechanism of biotin-dependent enzymes” Annu. Rev. Biochem. 58:195-221.

The Citric Acid Cycle


Chapter 10

References

  • Singer, T. P. and Johnson, M. K. (1985) “The prosthetic groups of succinate dehydrogenase: 30 years from discovery to identification” FEBS Lett. 190:189-196.

  • Velot, C., Mixon, M. B., Teige, M., and Srere, P. A. (1997) “Model of a quinary structure between Krebs TCA cycle enzymes: a model for metabolon” Biochemistry, 36:14271-14276.

  • Wolodko, W.T., Fraser, M. E., James, M. N. G., and Bridger, W. A. (1994) “The crystal structure of succinyl-CoA synthetase from E. coli” J. Biol. Chem. 269:10833-10890.

The Citric Acid Cycle


Chapter 10

References

  • Baldwinm J. E. and Krebs, H., (1981) “The evolution of metabolic cycles” Nature 291:381-382.

  • Reed, L. J., and Hackert, M. L. (1990) “Structure-functional relationships in dihydrolipoamide acyltransferases” J. Biol. Chem. 265:8971-8974.

  • Mattevi, A., Obmolova, G., Schulze, E., Kalk, K. H., Westphal, A. H., de Kok, A., and Hol, W. G. (1992) “Atomic structure of the cubic core of the pyruvate dehydrogenase multienzyme complex” Science 255:1544-1550.

The Citric Acid Cycle


Chapter 10

References

  • Perham, R. N. (1991) “Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: A paradigm in the design of a multifunctional protein, Biochemistry 30:8501-8512.

  • Patel, M. S., and Roche, T. E. (1990) “Molecular biology and biochemistry of pyruvate dehydrogenase complexes” FASEB J. 4:3224-3233.

  • Green, J. D., Perham, R. N., Ullrich, S. J., and Appella, E. (1992) “Conformational studies of the interdomain linker peptides in the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of E. coli” J. Biol. Chem. 267:23484-23488.

The Citric Acid Cycle


Chapter 10

References

  • Karpusas, M., Branchaud, B., and Remington, S. J. (1990) “Proposed mechanism for the condensation reaction of citrate synthase: 1.9 A structure of the ternary complex with oxaloacetate and carboxylmethyl coenzyme A” Biochemistry 29:2213-2219.

  • Lauble, H., Kennedy, M. C., Beinert, H., and Stout, C. D. (1992) “Crystal structures of aconitase with isocitrate and nitroisocitrate bound” Biochemistry 31:2735-2748.

  • Barnes, S. J., and Weitzman, P. D. (1986) “Organization of citric acid cycle enzymes into a multienzyme cluster” FEBS Lett. 201:267-270.

The Citric Acid Cycle


Chapter 10

The Citric Acid Cycle

The End


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