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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 carry 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 the 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 ONE
Enzyme: 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 of 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 3
Enzyme: 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 the enzyme 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 to the active oxidized disulfide form Lipoamide

(oxidant)
(reductant) (oxidized)

(reduced)

STEP 5
Enzyme: Dihydrolipoamide dehydrogenase Cofactor: NAD+
3rd substrate Oxidizes the FADH2 prosthetic group of E3 back to FAD regenerating the original holoenzyme and completing the catalytic cycle

Overview of Products and Substrates Step 1: Substrate 1: Pyruvate Product 1: CO2 Step 3: Substrate 2: Coenzyme A Product 2: Acetyl CoA Step 5: Substrate 3: NAD+ Product 3: NADH 3

The Citric Acid Enzymes:


-Localized in the mitochondria

Citric Acid Cycle


For 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 Acceptor Molecule Feeder Molecule

Release of First CO2

Release of Second CO2 Step 1 2 3 4 5 6 Substrate Acetyl-CoA Citrate Isocitrate -Ketoglutarate Succinyl CoA Succinate Reaction Type Condensation Dehydration Oxidation decarboxylation -Ketoglutarate Oxidative dehydrogenase complex decarboxylation Succinyl-CoA synthetase Substrate-level phosphorylation Succinate dehydrogenase Oxidation Complex Fumarase Hydration Malate dehydrogenase Oxidation GTP(ATP) per cycle: 1 NADH per cycle: 3 Enzyme Citrate synthase Aconitase Isocitrate dehydration Product HS-CoA, H+ NADH, CO2 NADH, CO2 GTP (or ATP), HSCoA QH2

7 Fumarate 8 Malate Cycles per Glucose: 2

NADH, H+ Location: mitochondria

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


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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 readdition 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 Aconitate

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- Stereospecific re-addition of H2O to cis-Aconitate forms 2R,3SIsocitrate. - Aconitase is a Metabolically Reversible reaction.

Citrate is a non-oxidizable tertiary alcohol.

Isocitrate is an oxidizable secondary alcohol.

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


decarboxylates by -elimination, releasing CO2, forming -Ketoglutarate.

Isocitrate + NAD+ -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 -elimination reaction. - Oxalosuccinate is the intermediate in this dehydrogenation reaction and is enzyme-bound - It is a -keto tricarboxylic acid

4. -Ketoglutarate Dehydrogenase Complex:


Ketoglutarate

Oxidizes and Decarboxylates -

-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**

-Ketoglutarate dehydrogenase complex

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


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

- conserves energy of a thioester to form GTP(a nucleotide diphosphate) - Once called T k n - 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 phosphoryl group to GDP forming GTP.
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6. Succinate Dehydrogenase Complex:


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

Catalyzes the oxidation of succinate to fumarate,

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, and 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 stereospecific 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:
molecule of NADH.

Oxidation of malate to regenerate oxaloacetate, with formation of a

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


- Uses NAD+ k n - This NAD n O , n n - At equilibrium the reaction lies far to the LEFT; the level of L-Malate is much higher than the level of 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 associated with the production of about 10 molecules of ATP. Reaction Isocitrate dehydrogenase (Step 3) -Ketoglutarate dehydrogenase complex (Step 4) Succinyl-CoA synthetase (Step 5) Succinate dehydrogenase complex ( Step 6) Malate dehydrogenase (Step 8) Total Energy-yielding product NADH NADH GTP or ATP QH2 NADH ATP equivalents 2.5 2.5 1.0 1.5 2.5 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 (Nucleotide Triphosphate) --NADH and QH2 are reoxidized in the Electron Transport Scheme and produce much more ATP by Oxidative Phosphorylation. Glycolysis converts glucose to 2 molecules of pyruvate with a net gain of 2 molecules of ATP. There are 2 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 NADH molecules, which correspond to about 5 additional molecules of ATP.

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Fate of Acetyl-CoAs 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 become 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 the same carbon atoms that are lost as CO2.

o The carbon balance in the overall reaction pathway: for each 2-C group from acetyl CoA that enters the cycle, 2 carbon atoms are released during 1 complete turn of the cycle. o The 2 C atoms of acetyl CoA become half of the symmetric 4 carbon dicarboxylic acid (succinate) in the 5th step of the cycle. o The 2 halves of this symmetric molecule are chemically equivalent so carbons arising from acetyl CoA become 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 subsequent 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 -ketoglutarate in the blood. How n y n n n y n -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 ketoglutarate dehydrogenase complex. A deficiency of TP decreases the activities of these enzyme complexes. Decreasing the conversion of pyruvate to acetyl CoA and of -ketoglutarate to succinyl CoA causes accumulation of pyruvate and -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 excretion n n bn y n -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 -ketoglutarate, which could lead to excretion of these molecules. 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 -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 n y y n n -ketoglutarate to succinyl CoA is the -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 -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 irreversible 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 -ketoglutarate to succinyl CoA 1.

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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 -ketoglutarate is oxidatively decarboxylated by the -ketoglutarate dehydrogenase complex 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 the inner mitochondrial membrane). How are these catabolic pathways connected despite being located in different cell 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 agent 2. ________ 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 known 8. ________ 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 succinyl CoA 10. ________ Two moles of coenzyme A are consumed per mole of pyruvate catabolized to acetyl CoA, which is 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 by substitution of a single functional group. 4 ________ T -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 acid cycle 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 hydrogens 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 intermediates are continually recycled. However, sufficient amounts of oxaloacetate must be present to allow appropriate rates 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, in symport 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 -ketoglutarate dehydrogenase complex. 12. True. Glycolysis produces 2 ATP per mole of glucose. From the two moles of pyruvate produced by glycolysis, 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 molecule by n n by n nn T b nw then 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 as GTP (or ATP) is formed.

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