生物化学精品课程 Chapter 10 The Citric Acid Cycle The common pathway leading to complete oxidation of carbohydrates, fatty acids, and amino acids to CO 2. A pathway

生物化学精品课程

Chapter 10Chapter 10

The Citric Acid Cycle

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

A pathway providing many precursors for biosynthesis.


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

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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.


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

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Mitochondria is the major site forfuel oxidation to generate ATP.


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

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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 dehydrogenase complex 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）



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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+.


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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).



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The oxidative decarboxylation of pyruvatein mitochondria: producing acetyl-CoA and CO2.



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E3

hydroxyethyl-TPP

pyruvate CO2acetyl-CoA

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)

A model of the E. coli pyruvate dehydrognase complex showing the

three kinds of enzymes and the flexible lipoamide arms covalently

attached to E2.


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

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


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


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

decarboxylase.


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Dihydrolipoyl

The lipoyllysyl group serves as the electron and acetyl carriers


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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!


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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).


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4. The acetyl group (carried by CoA) is completely oxidized to CO2 via 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.


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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.


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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.


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Step 4 α-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 thioester bond 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 );


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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.


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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 8 Oxaloacetate is regenerated by the oxidation of

L-malate; this reaction is catalyzed by malate

dehydrogenase with two electrons collected by NAD+.


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The aldol condensation between acetyl-CoA and oxaloacetate forms citrate


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Citrate synthase beforeand after binding to

oxaloacetate

Oxaloacetate

Carboxylmethyl-CoA


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4Fe-4S cubic array: each Fe is bonded to three inorganic S

and a cysteine sulfur atom (except one)

Citrate is converted to isocitrate via dehydration followed by a Hydration （水合作用） step.


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Isocitrate is converted to -ketoglutarate via an oxidative decarboxylation step, generating NADH

CO2.

The first oxidation step


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The second oxidation step

TPP lipoate ， FAD

(E1, E2, E3)

The-ketoglutarate dehydrogenase complex closely resembles the pyruvate dehyrogenase

complex in structure and function


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Succinyl-CoA synthetasecatalyzes the substrate-level

phosphorylation of ADP.


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Succinyl-CoA Synthetase from E. coli

Coenzyme A

His246-Pi

The power helices


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The third oxidation step

(An enzyme bound tothe inner membrane of mitochondria)


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(a stereospecific enzyme)


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(The fourth oxidationStep in the cycle)

Oxaloacetate is regenerated at the end


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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.


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


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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.


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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+.


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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.


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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+).


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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.


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

The isocitrate dehydrogenase andthe isocitrate lyase are coordinately

regulated.


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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生物化学精品课程 Chapter 10 The Citric Acid Cycle The common pathway leading to complete oxidation of carbohydrates, fatty acids, and amino acids to CO 2. A pathway