CH 13 Review

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Chapter 13 Chem. 555/Biol. 545 PRINCIPLES OF BIOCHEMISTRY

THE CITRIC ACID CYCLE: (Krebs/Tricarboxylic [TCA] Cycle)

LECTURE OBJECTIVES

1. Understand the central position of the PDH reaction in overall metabolism.

Pyruvate Dehydrogenase (PDH) is a polyfunctional enzyme complex that utilizes FIVE different cofactors to car

out oxidative decarboxylation of Pyruvate.

**Commited step in Pyruvate Catabolism**

2. Know the sequence of reactions involved in the conversion of pyruvate to acetyl CoA: the structure of t

pyruvate dehydrogenase (PDH) complex; the role of each enzyme and coenzyme in its action.

PDH Complex: consists of 3 enzymes and 5 cofactors

• 3 enzymes

– E1 = Pyruvate dehydrogenase

– E2 = Dihydrolipoamide acetyltransferase

– E3 = Dihydrolipoamide dehydrogenase

• 5 Cofactors

– Thiamine pyrophosphate

– FAD

– Lipoic acid

– NAD+

– Coenzyme A (SH-CoA)

STEP ONEEnzyme: Pyruvate dehydrogenase

Cofactor: Thiamine PP (TPP)

- Decarboxylates Pyruvate

- The C atom flanked by the S and N acts as a carbanion which attacks the carbonyl C o

Pyruvate leads to loss of CO2forming 1st

product in overall reaction

Hydroxyethylthiamine pyrophosphate (HETPP): First product of overall reaction

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- It is the acetaldehyde-adduct of the cofactor TPP

- It is decarboxylated Pyruvate

STEP 2

Enzyme: Pyruvate dehydrogenase

Cofactor: Lipoamide

- Prosthetic group- Oxidizing agent that converts HETPP (the acetaldehyde TPP adduct) Acetyl dibydrolipoamide

STEP 3Enzyme: Dihydrolipoamide acetyltransferase

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Cofactor: Coenzyme A

- Second Substrate

The disulfhydryl (reduced) form of Lipoamide must be reoxidized back to the disulfide (oxidized) form for tenzyme complex to continue operating.

STEP 4

Enzyme: Dihydrolipoamide dehydrogenase

- The FAD group of E3 Oxidizes the reduced Lipamide of E2 forming FADH2

Cofactor: FAD

- Prosthetic group that serves as the OXIDANT to convert reduced Dihydrolipoamide back

the active oxidized disulfide form Lipoamide

STEP 5Enzyme: Dihydrolipoamide dehydrogenase

Cofactor: NAD+

- 3rd substrate

- Oxidizes the FADH2 prosthetic group of E3 back to FAD regenerating the original holoenzyme acompleting the catalytic cycle

Overview of Products and Substrates

Step 1: Step 3: Step 5:

Substrate 1: Pyruvate Substrate 2: Coenzyme A Substrate 3: NAD+

Product 1: CO2 Product 2: Acetyl CoA Product 3: NADH

(reductant) (oxidized)

(oxidant) (reduced)

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The Citric Acid Enzymes:-Localized in the mitochondria

Citric Acid CycleFor each acetyl group that enters the pathway:

- 2 molecules of CO2 are released - mobile coenzymes NAD+

and Q are reduced

- 1 molecule of GDP (or ADP) are phosphorylated - oxaloacetate is re-formed

Step Substrate Enzyme Reaction Type Product

1 Acetyl-CoA Citrate synthase Condensation HS-CoA, H+

2 Citrate Aconitase Dehydration

3 Isocitrate Isocitrate dehydration Oxidation

decarboxylation

NADH, CO2

4 α-Ketoglutarate α-Ketoglutarate

dehydrogenase complex

Oxidative

decarboxylation

NADH, CO2

5 Succinyl CoA Succinyl-CoA synthetase Substrate-level

phosphorylation

GTP (or ATP), HS-

CoA6 Succinate Succinate dehydrogenase

Complex

Oxidation QH2

7 Fumarate Fumarase Hydration

8 Malate Malate dehydrogenase Oxidation NADH, H+

Cycles per Glucose: 2 GTP(ATP) per cycle: 1 NADH per cycle: 3 Location: mitochondria

1. Citrate Synthase: condensation of Acetyl CoA with Oxaloacetate to form Citrate

Release of

Second CO2

Release of First CO2

Acceptor Molecule Feeder Molecule

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Acetyl CoA + Oxaloacetate + H2O Citrate + HS-CoA + H+

First Metabolically Irreversible Reaction

- Driven by the high free energy of the thioester of Acetyl CoA

Only Reaction in TCA Cycle in which C-C bon formation occurs.

Acceptor Molecule: Oxaloacetate (4-C)Feeder Molecule: Acetyl CoA (2-C)

alcohol that cannot be oxidized under biological conditions

2. Aconitase: first causes the removal of water from Citrate to form cis-aconitate. Stereo specific r

addition of water forms Isocitrate.

Citrate↔ Isocitrate

**NOTE**

Primary alcohol: (one carbon bound)

- Oxidizes to a carboxylic acid or aldehye

Secondary alcohol: (two carbon bound)

- Oxidizes to a ketone

Tertiary alcohol: (three carbons bound)

- Not easily oxidized

- Aconitase removes H2O from Citrate to form the C=C bond of ci

Aconitate

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- Stereospecific re-addition of H2O to cis-Aconitate forms 2 R,3S

Isocitrate.

- Aconitase is a Metabolically Reversible reaction.

3. Isocitrate Dehydrogenase: oxidizes Isocitrate to an unstable -keto form that spontaneous

decarboxylates by β-elimination, releasing CO2, forming

-Ketoglutarate.

Isocitrate + NAD+ α-Ketoglutarate + NADH + CO2

**Second METABOLICALLY IRREVERSIBLE reaction of the TCA Cycle.**

**1st

of 4 Oxidation-Reduction Reactions of the TCA Cycle**

forming an -keto group.

#2. Oxalosuccinate spontaneously converts to -KG releasing CO2 via a β-elimination reaction.- Oxalosuccinate is the intermediate in this dehydrogenation reaction and is enzyme-bound

- It is a -keto tricarboxylic acid

Citrate is a

non- oxidizable tertiary alcohol .

Isocitrate is an

oxidizable

secondary alcohol.

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4. α-Ketoglutarate Dehydrogenase Complex: Oxidizes and Decarboxylates

Ketoglutarate

α-Ketoglutarate + HS-CoA + NAD+ Succinyl CoA + NADH + CO2

- Multi-enzyme complex similar to Pyruvate dehydrogenase complex

- -Ketoglutarate is an -Keto acid

- Products: CO2, NADH and Succinyl CoA.

- Succinyl CoA: high energy Thioester that participates in substrate-level phosphorylation in the next reaction

**Second Oxidative Decarboxylation**

**Third Metabolically Irreversible Reacion**

5. Succinyl- CoA Synthetase: Catalyzes the conversion of succinyl CoA to succinate

Succinyl CoA + GDP (or ADP) + Pi ↔ Succinate + GTP (or ATP) + HS-CoA

α - Ketoglutarate


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- conserves energy of a

thioester to form GTP(a

nucleotide diphosphate)

- Once called

“Tkn”

- Substrate-level

phosphorylation

- Pi becomes

incorporated during the

energy transfer to

convert GDP to GTP

Proposed Mechanism of

Succinyl-CoA Synthase

Step 1 – Phosphate displaces SH-CoA

to form the mixed acid anhydride,succinyl phosphate.

Step 2 – The phosphoryl group is

transferred to a histidine residue of the

enzyme forming a relatively stable

covalent phosphoenzyme intermediate.

Step 3 – Succinate is released, and the

phosphoenzyme transfers its phosphory

group to GDP forming GTP.

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6. Succinate Dehydrogenase Complex: Catalyzes the oxidation of succinate to fumara

forming a C=C with the loss of 2 protons and 2 electrons.

Succinate + Q↔ Fumarate + QH2

Succinate has two planes of symmetry. It is NOT a pro-chiral molecule and

C-1 can NOT be distinguished from C-4.

- A “prochiral” molecule is achiral but can be converted to a chiral molecule in a

single step.

- Contains an FAD prosthetic group that serves as the primary oxidizing agent.

- The FAD prosthetic group then transfers the electrons through Fe-S cluster to Q, forming QH2, a

mobile lipophilic redox agent.

Characteristic features of FAD-dependent dehydrogenases are:

a) the substrate has

adjacent – CH2 groups, an

b) removal ’

yields a trans double bond

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• Succinate Dehydrogenase is a complex of several polypeptides.

• It contains FAD as a prosthetic group, as well as Iron-Sulfur (Fe-S

clusters.

• The SDH Complex is located in the Inner Mitochondrial Membrane

7. Fumerase: Catalyzes the near-equilibrium conversion of fumarate to malate through the stereospecif

trans addition of water to the double bond of fumarate.

Fumarate + H2O↔ L-Malate

- Causes stereospecific addition of H2O to the trans double bond of Fumarate forming L-Malate

8. Malate Dehydrogenase: Oxidation of malate to regenerate oxaloacetate, with formation o

molecule of NADH.

L-Malate + NAD+ ↔ Oxaloacetate + NADH + H

+

- Uses NAD+ kn

- This NAD n O, nn “ ” - At equilibrium the reaction lies far to the LEFT; the level of L-Malate is much higher than the level

Oxaloacetate.

- Because of this, Oxaloacetate is the limiting substrate in the TCA Cycle.

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IV. Energy yield of ATP per acetyl-CoA unit.

Net reaction of the Citric Acid Cycle:

Acetyl CoA + 3 NAD+

+ Q + GDP + Pi + 2 H2O HS-CoA + 3 NADH + QH2 + GTP + 2 CO2 + 2 H+

The complete oxidation of 1 molecule of acetyl CoA by the citric acid cycle and subsequent reactions is associatwith the production of about 10 molecules of ATP.

Reaction Energy-yielding product ATP equivalents

Isocitrate dehydrogenase (Step 3) NADH 2.5

α-Ketoglutarate dehydrogenase complex (Step 4) NADH 2.5

Succinyl-CoA synthetase (Step 5) GTP or ATP 1.0

Succinate dehydrogenase complex ( Step 6) QH2 1.5

Malate dehydrogenase (Step 8) NADH 2.5

Total 10.0

The TCA cycle- is a mechanism for oxidizing Acetyl CoA to CO2 using NAD

+and Q as oxidants

- Does not cause NET degradation of any cycle intermediates

--Energy is conserved in the form of Reduced Coenzymes: 3 NADH, 1 QH2 and 1 GTP (Nucleoti

Triphosphate)

--NADH and QH2 are reoxidized in the Electron Transport Scheme and produce much more ATP

Oxidative Phosphorylation.

Glycolysis converts glucose to 2 molecules of pyruvate with a net gain of 2 molecules of ATP. There are

molecules of NADH produced in the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. = 7 ATP

The conversion of both pyruvate molecules to acetyl CoA by the pyruvate dehydrogenase complex yields 2 NADmolecules, which correspond to about 5 additional molecules of ATP.

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Fate of Acetyl-CoA’s carbon Units

Carbon atoms from Acetyl CoA (red) are not lost as CO2 in the first turn of the cycle.

At Succinate, because of its 2 planes of symmetry, atoms from Acetyl CoA becom

randomized.

Only in the second cycle do labeled C-atoms from Acetyl CoA become oxidized to CO2

The 2 C atoms that enter the cycle as the acetyl group on acetyl CoA are not t

same carbon atoms that are lost as CO2.

o The carbon balance in the overall reaction pathway: fo

each 2-C group from acetyl CoA that enters the cycle,

carbon atoms are released during 1 complete turn of th

cycle.

o The 2 C atoms of acetyl CoA become half of thsymmetric 4 carbon dicarboxylic acid (succinate) in the 5

step of the cycle.

o The 2 halves of this symmetric molecule are chemicall

equivalent so carbons arising from acetyl CoA becom

evenly distributed in molecules formed from succinate.

For each 2-C acetyl-CoA unit that enters the TCA cycle:-2 CO2 molecules generated - b ’ y

-3 NADH (+ 3 H+), 1 QH2, and 1 GTP (equiv. To 1 ATP) generated.

The ’ y-CoA are not oxidized in the TCA cycle, but are transferred to redox cofactors for subseque

oxidation in the electron-transport scheme (ETS).

Regulation of the Citric Acid Cycle

The Rate of the Cycle is controlled at 4 sites:

1) The Pyruvate Dehydrogenase Complex, which controls the supply of Acetyl CoA.

2) Citrate Synthase

3) Isocitrate Dehydrogenase

4) The -Ketoglutarate Dehydrogenase Complex

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The principal allosteric inhibitors are NADH and ATP, both of which signal energy excess.

1) Regulation of PDH Complex

A. By Allosteric Modulation

Increased levels of Acetyl CoA and NADH inhibit E2 & E3. Feedback Regulation

Increased levels of CoA-SH and NAD+ activate E2 & E3. Feed Forward Regulation

B. By Covalent Modification (Mammalian PDH Complex)

Phosphorylation INACTIVATES E1.Dephosphorylation ACTIVATES E1.

The Substrates make the complex ACTIVE by inhibiting PDH Kinase: NAD+,

HS-CoA, ADP and Pyruvate

The Products INACTIVATE the complex by activating PDH Kinase: NADH

and Acetyl CoA

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Overview of Regulation sights 2-4

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Suggested Problems:

5. The disease beriberi, which results from a dietary deficiency of vitamin B1 (thiamine), is characterized by

n n y, w n y n α-ketoglutarate in the blood. How

ny n n n y n α-ketoglutarate?

Thiamine is the precursor of the coenzyme thiamine pyrophosphate (TPP), which is found in two enzyme

complexes associated with the citric acid cycle: the pyruvate dehydrogenase complex and the α-

ketoglutarate dehydrogenase complex. A deficiency of TP decreases the activities of these enzyme

complexes. Decreasing the conversion of pyruvate to acetyl CoA and of α-ketoglutarate to succinyl CoAcauses accumulation of pyruvate and α-ketoglutarate.

8. A deficiency of a citric acid cycle enzyme in both mitochondria and the cytosol of some tissues (e.g. blood

lymphocytes) results in severe neurological abnormalities in newborns. The disease is characterized by excretio

n n bny n α-ketoglutarate, succinate and fumarate. What enzyme deficiency

would lead to these symptoms?

A deficiency in the citric acid cycle enzyme fumarase would result in abnormally high concentrations of

fumarate and prior cycle intermediates including succinate and α-ketoglutarate, which could lead to

excretion of these molecules.

1. The oxidation of pyruvate to acetyl CoA and CO2 requires the cofactors coenzyme A, NAD+, lipoate, FAD

and TPP.

2. Four dicarboxylic acid intermediates of the citric acid cycle are succinate, fumarate, malate and

oxaloacetate. 3. The conversion of α-ketoglutarate also produces CO2. This is an oxidative process in which the

actual oxidant is NAD+.

4. Transformation of pyruvate to acetyl CoA is catalyzed by the pyruvate dehydrogenase complex [This

complex contains multiple copies of pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase

(E2), and dibydrolipoamide dehydrogenase (E3).]

5. The enzyme that catalyzes the first step of the citric acid cycle is citrate synthase 6. T ny y nn α-ketoglutarate to succinyl CoA is the α-ketoglutarate

dehydrogenase complex. 7. The only step in the citric acid cycle that directly produces an energy-rich phosphoanhydride involves the

conversion of succinyl CoA to succinate

8. There are a total of five (including the conversion of pyruvate to acetyl CoA oxidation-reduction steps in

the catabolism of pyruvate via the citric acid cycle. The specific reduced coenzymes formed are NADH and

QH2 9. The competitive inhibitor malonate slows the citric acid cycle and causes succinate to accumulate.

10. Of the 8 steps in the citric acid cycle, the number of steps that produce CO2 is two.

11. Certain citric acid cycle intermediates serve as biosynthetic precursors of other materials. Two such

intermediates are α-ketoglutarate and succinyl CoA (as well as citrate and oxaloacetate) 12. A negative modulator involved in regulating each of the enzymes that catalyze the 3 metabolically irreversib

reactions of the citric acid cycle is NADH

13. The intermediate L-malate is formed by the hydration of fumarate in the cycle.14. The second CO2 producing step in the citric acid cycle involves the conversion of α-ketoglutarate to succin

CoA

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1. What is coenzyme A and what is its function in the oxidation of pyruvate via the citric acid cycle?

Coenzyme A is composed of an ADP moety, an esterified phosphate, a pantothenic acid unit, and a

mercaptoethylamine unit. Coenzyme A functions as a carrier of acyl groups such as the acetyl group

formed when pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex, and the

succinyl group formed when α-ketoglutarate is oxidatively decarboxylated by the α-ketoglutarate


3. In eukaryotes, pyruvate is produced from glucose by glycolysis in the cytosol, but the enzymes of the citric

acid cycle are in the mitochondrial matrix (except for succinate dehydrogenase, which is part of Complex II in thinner mitochondrial membrane). How are these catabolic pathways connected despite being located in differentcell compartments?

Pyruvate, produced in the cytosol by glycolysis, enters mitochondria through porins in the outer

membrane and by the aid of pyruvate translocase in the inner membrane. In the mitochondrial matrix,

pyruvate is converted to acetyl CoA, a substrate for the citric acid cycle.

True/False

1. ________ FAD is used in the citric acid cycle as an oxidizing agent2. ________ The final steps in the oxidation of glucose to CO2 occur in the citric acid cycle.

3. ________ Massive amounts of all intermediates of the citric acid cycle must be present for its efficient

operation.4. ________ In eukaryotes, the enzymes that catalyze the reactions of the citric acid cycle are found in the

cytosol.

6. ________ Pyruvate is produced in the eukaryotic cytosol but enters the mitochondrial matrix unaided.

7. ________ The eukaryotic pyruvate dehydrogenase complex is the largest multienzyme complex known8. ________ Intermediates of the citric acid cycle that are used for anabolic (synthetic) needs of the cell are

replenished by anaplerotic reactions.

9. ________ Porphyrin biosynthesis could potentially interfere with the citric acid cycle by depleting succinylCoA

10. ________ Two moles of coenzyme A are consumed per mole of pyruvate catabolized to acetyl CoA, which

subsequently consumed in the citric acid cycle.11. ________ The citric acid is controlled entirely by the availability of acetyl CoA and cycle intermediates.

12. ________ Per mole of glucose, the citric acid cycle produces as many high-energy phosphate molecules by

substrate level phosphorylation as does glycoltic catabolism of glucose to pyruvate.

13. ________ A prochiral molecule is a symmetrical molecule that can be converted to a chiral molecule bysubstitution of a single functional group.

4 ________ T α-ketoglutarate dehydrogenase complex is very much like the pyruvate dehydrogenase

complex.

15. ________ The reaction catalyzed by the succinate dehydrogenase complex is the only step in the citric acidcycle that involves substrate level phosphorylation.

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1. True. FAD is a prosthetic group of an enzyme of the succinate dehydrogenase complex that accepts hydroge

form succinate and immediately transfers them to coenzyme Q, the mobile carrier of reducing power within the

inner mitochondrial membrane.

2. True. The citric acid cycle generages 6 molecules of CO2 for each molecule of glucose catabolized.3. False. Your text points out that the cycle is a multistep catalyst. Since the process is cyclic, the intermediate

are continually recycled. However, sufficient amounts of oxaloacetate must be present to allow appropriate rate

of acetyl CoA uptake.4. False. There are 2 six-carbon intermediates, 1 five-carbon intermediate, and 5 four-carbon intermediates.

5. False. They are located in the mitochondria.6. False. Pyruvate does pas freely through porins in the outer mitochondrial membrane, but its passage, insymport with H+, through the inner mitochondrial membrane is aided by pyruvate translocase.

7. True. It is several times larger than a ribosome.

8. True.9. True. Succinyl CoA condenses with glycine to initiate porphyrin biosynthesis, but succinyl CoA can be

replenished by degradation of some amino acids.

10. False. Although conenzyme A is required in 2 different steps as pyruvate is catabolized, two moles of

coenzyme A are formed as products in the citric acid cycle, so there s no net consumption of coenzyme A.11. False. In addition to the availability of acetyl CoA and oxaloacetate to initiate citrate formation, control is

exercised through allosteric modulators and their effects on the cycle enzymes citrate synthase, isocitrate

dehydrogenase, n α-ketoglutarate dehydrogenase complex.12. True. Glycolysis produces 2 ATP per mole of glucose. From the two moles of pyruvate produced byglycolysis, the citric acid cycle produces 2 moles of GTP (or ATP)

13. True. The prochiral molecule contains a carbon of the type Caacd which can be converted to a chiral molecu

by n n α by n nn T bn wthen be Cabcd

14. True. Both are complexes of several different enzymes and utilize the same cofactors (TPP, lipoamide, FAD

CoASH, and NAD+)

15. False. It is the step catalyzed by succinyl CoA synthase that results in substrate level phosphorylation asGTP (or ATP) is formed.

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