Chapter 16

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

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

into three stages• Stage I All the fuel molecules are oxidized to generat

e 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.

Mitochondria is the major site forfuel oxidation to generate ATP.

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

hydrogenase complex• Pyruvate is first transported into mitochondria via a specific tr

ansporter 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 (TPP) of pyruvate dehydrogenase (E1), forming hydroxyethyl-TPP.

• 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 (E2).

• The lipoyllysyl group serves as both electron and acetyl carriers.

• The acetyl group is then transferred (still catalyzed by E2) from acetyllipoamide to CoA-SH,forming acetyl-CoA.

• The oxidized lipoamide group is then regenerated by the action of dihydrolipoyl dehydrogenase (E3), with electrons collected by FAD and then by NAD+.

• 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 -keto acids, pyruvate dehydrogenase complex, -ketoglutarate dehydrogenase complex and branched chain -keto acid dehydrogenase complex show remarkable structure and function relatedness (all have identical E3, similar E1 and E2).

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

Electron micrograph of pyruvate dehydrogenase complexes from E. coli

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

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)

pyruvate

E2

E3

hydroxyethyl-TPP

CO2 acetyl-CoA

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 hollowcube.

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

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

Dihydrolipoyl

The lipoyllysyl group serves as the lectron and acetyl carriers

Coenzyme A (CoA-SH): discovered in 1945 by Lipmann, one of the “carrier molecules”, deliver activated acyl groups (with 2-24Carbons) for degradation or biosynthesis.

3. The complete oxidation of pyruvate in animal tissues was proposed to under

go via a cyclic pathway• O2 consumption and pyruvate oxidation in minced muscle t

issues were found to be stimulated by some four-carbon dicarboxylic acids (Fumarate, succinate, malate and oxaloacetate, five-carbon dicarboxylic acid (-ketoglutarate ), or six-carbon tricarboxylic acids (citrate, isocitrate, cis-aconitate).

• A small amount of any of these organic acids stimulates mmany foldsany folds of pyruvate oxidation!

• Malonate inhibits pyruvate oxidation regardlessregardless of which active organic acid is added!

• 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).

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

e 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 citricacid cycle

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.

• 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 -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.

• Step 4 -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 -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);

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 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.

Step 7 Fumarate is hydrated to L-malate by the action of fumarase; 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 OxaloacetateOxaloacetate is regenerated by the oxidation of L-malate; this reaction is catalyzed by malate dehydrogenase with two electrons collected by NAD+.

The aldol condensation between acetyl-CoA and oxaloacetate forms citrate

Citrate synthase beforeand after binding to oxaloacetate

Oxaloacetate

Carboxylmethyl-CoA

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 step.

Isocitrate is converted to -ketoglutarate via an oxidative decarboxylation step, generating NADH CO2.

The first oxidation step

TPP lipoate FAD

(E1, E2, E3)

The-ketoglutarate dehydrogenase complex closely resembles the pyruvate dehyrogenase complex in structure and function

The second oxidation step

Succinyl-CoA synthetasecatalyzes the substrate-levelphosphorylation of ADP.

Succinyl-CoASynthetase fromE. coli

His246-Pi

Coenzyme A

The power helices

(An enzyme bound tothe inner membrane of mitochondria)

The third oxidation step

(a stereospecific enzyme)

(The fourth oxidationStep in the cycle)

Oxaloacetate is regenerated at the end

5. 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.

6. The citric acid intermediates are important sources for biosynthetic pre

cursors • The citric acid cycle is the hub of intermediary meta

bolism serving both the catabolic and anabolic processes (thus an amphibolic pathway).

• 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 by anaplerotic reactions when consumed by biosynthesis.

• The most common anaplerotic reactions covert either pyruvate or phosphoenolpyruvate to oxaloacetate or malate.

• Pyruvate carboxylase catalyzes the carboxylation of pyruvate using covalently bound biotin coenzyme.

• The activated CO2 is first attached to an ureido N on biotin and then transferred to the ionized enolate form of pyruvate, producing oxaloacetate.

• The long flexible arm of biotin switches between two active site of the pyruvate carboxylase (one for attaching a HCO3

- to biotin and the other for transferring the carboxyl group to pyruvate).

• Acetyl-CoA is a positive allosteric modulator for the carboxylase.

• Some anaerobic bacteria, lacking the -ketoglutarate dehydrogenase enzyme, make biosynthetic precursors via the incomplete citric acid cycle; could be an early evolution stage of the citric acid cycle.

Active site 1

Active site 2

The long flexiblearm of biotin switches betweentwo active sites of pyruvatecarboxylase

Incomplete citric acid cycle has beenfound in some anaerobic bacteria

7. Net conversion of acetate to carbohydrates is allowed via the glyoxylate cyc

le • There is no net conversion of acetate (also from fatty acid

s and amino acids) to any of the citric acid cycle intermediate, thus neither to carbohydrates.

• Net conversion of acetate to four-carbon citric acid cycle intermediates occurs via the glyoxylate cycle, found in plants, certain invertebrates, and some microorganisms (including E. coli and yeast).

• The glyoxylate cycle shares three steps and bypasses the rest, including the two decarboxylation steps, of the citric acid cycle.

• Two acetyl-CoA molecules enter each glyoxylate cycle with a net production of one succinate.

• Isocitrate lyase and malate synthase are the two bypassing enzymes, converting isocitrate to malate via the glyoxylate intermediate, releasing a succinate on the way.

The glyoxylate cycle

8. Conversion of fatty acids to glucose (in germinating seeds) occurs in three i

ntracellular compartments• Fatty acids are first degraded into acetyl-CoA, whi

ch is in turn converted to succinate via the glyoxylate cycle in glyoxysomes.

• Succinate is transported to mitochondria and is converted to malate there via the citric acid cycle.

• Malate is then transported out of mitochondria and is converted to glucose in the cytosol.

Fatty acids in germinating seeds areconverted to glucose via three compartments

9. The pyruvate dehydrogenase complex in vertebrates is regulated alloseterica

lly and covalently• The formation of acetyl-CoA from pyruvate is a ke

y 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 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.

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

steps • Citrate synthase, isocitrate dehydrogenase and -ket

oglutarate 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+).

11. The partitioning of isocitrate, between the citric acid and glyoxylate cycles

is coordinately regulated • The activity of the E. coli isocitrate dehydrogenase is inh

ibited 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 isocitrate dehydrogenase andthe isocitrate lyaseare coordinatelyregulated

Summary• Pyruvate is converted to acetyl-CoA by the action of p

yruvate 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.

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

• 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 cycle and citric acid cycle via a coordinately regulation of the isocitrate dehydrogenase and isocitrate lyase.

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