CITRIC ACID CYCLE Student Edition 11/8/13 version Pharm. 304 Biochemistry Fall 2014 Dr. Brad...

CITRIC ACID CYCLEStudent Edition 11/8/13 version

Pharm. 304 Biochemistry

Fall 2014

Dr. Brad Chazotte 213 Maddox Hall

chazotte@campbell.eduWeb Site:

http://www.campbell.edu/faculty/chazotte

Original material only ©2002-14 B. Chazotte

Goals• Learn the Citric Acid Cycle sequence, enzymes, intermediates, products,

and control mechanisms.

• Learn the different stages of cellular respiration.

• Know that the citric acid cycle involves the oxidation of 2-carbon units.

• Be familiar with the function of the pyruvate dehydrogenase complex, its reaction types, general structure, and control mechanisms.

• Understand how degradative reactions provide cycle intermediates.

• Be familiar with role of the cycle in providing biosynthetic precursors.

• Understand the role of anaplerotic reactions

Do NOT memorize specific enzyme mechanisms

Complete Oxidation to Molecular OxygenGlucose Note: 1 cal =4.184J

C6H12O6 + 6 O2 6 CO2 + 6 H2O G° ’=-2823 kJ mole-1

Broken down into the half reactions:

C6H12O6 + 6 H2O 6CO2 + 24H+ + 24 e-

6 O2 + + 24H+ + 24 e- 12 H2O

Palmitic Acid

Palmitoyl-CoA + 23O2 + 131 Pi + 131 ADP CoA + 16CO2 + 146 H2O +131 ATP

Palmitic Acid + 23 O2 16 CO2 + 16 H2O G°’= -9790.5 kJ mole-1

129 ADP + 129Pi 129 ATP + 129 H2O G°’= +3941 kJ mole-1

129 ATP is the next yield since 2 ATP are needed to form palmitoyl-CoA from palmitic acid. To Form 1 ATP G°’= +30.54 kJ mole-1 = 7.3 kcal mole-1

Citric Acid Cycle

Cellular Respiration Review

Citric Acid Cycle

Lehninger 2000 Fig 16.1a

Stage 1 of Cellular Respiration

Citric Acid Cycle

Lehninger 2000 Fig 16.1b

Stage 2 of Cellular Respiration

Citric Acid Cycle

Lehninger 2000 Fig 16.1c

Stage 3 of Cellular Respiration

Citric Acid Cycle

Overviewof the Citric Acid Cycle

Citric Acid Cycle

• The central metabolic hub of the cell

• The gateway to the aerobic metabolism for any molecule that can be converted into an acetyl group or a dicarboxylic acid.

Berg, Tymoczko & Stryer, 2012 Chap. 17 p.497

Coenzyme A

Citric Acid Cycle

Acetyl Coenzyme A

• Acetyl CoA is the “fuel” for the citric acid cycle

• Formed from the breakdown of glycogen, fats, and many amino acids.

• A high energy compound G° = -31 kJ mol-1

Lehninger 2000 Fig 16.3

Coenzyme A components

Citric Acid Cycle

Berg, Tymoczko & Stryer, 2012 Fig. 17.1

Mitochondrion Electron Micrograph

Citric Acid Cycle

Berg, Tymoczko & Stryer, 2012 Fig. 17.2

Citric Acid Cycle: Schematic Overview

Citric Acid CycleHorton 2012 Fig. 13.5

succinate

-ketoglutarate

oxaloacetate

acetyl group

citrate

isocitrate

A B

Berg, Tymoczko & Stryer, 2012 Fig. 17.3

Cellular Respiration Schematic

Citric Acid Cycle

“The function of the citric acid cycle is the harvesting of high energy electrons from carbon fuels”.

Enzymes of the Citric Acid Cycle

1. Citrate synthase

2. Aconitase

3. Isocitrate dehydrogenase

4. -ketoglutarate dehydrogenase

5. Succinyl-CoA synthetase

6. Succinate dehydrogenase

7. Fumarase

8. Malate dehydrogenase

Citric Acid Cycle

Intermediates of the Citric Acid Cycle

1. oxaloacetate (4C)

2. citrate (6C)

3. cis-aconitate (6C)

4. isocitrate (6C)

5. -ketoglutarate (5C)

6. succinyl-CoA (4C)

7. succinate (4C)

8. fumarate (4C)

9. malate (4C)

Citric Acid Cycle

“Products” of the Citric Acid Cycle

Three (3) Hydride Ions (H-), that is six

(6) electrons are produced in the form of:

3 NADH (from isocitrate dehydrogenase,

-ketoglutarate dehydrogenase, & malate dehydrogenase)

1 FADH2 (from succinate dehydrogenase)

These electron carriers donate to electron transport which in turn drives oxidative phosphorylation to produce ATP

1 GTP (or ATP)(from succinyl CoA synthetase, a substrate-level phosphorylation)

2 CO2 (at isocitrate dehydrogenase & -ketoglutarate dehydrogenase)

Citric Acid Cycle

Horton 2002 Fig12.6

Berg, Tymoczko & Stryer, 2012 Fig. 17.15

Citric Acid (Krebs) Cycle

Citric Acid Cycle

Berg, Tymoczko & Stryer, 2012 Table 17.2

Citric Acid (Krebs) Cycle Rx List

Citric Acid Cycle

Oxidation of Two Carbon Units[Citric Acid Cycle]

Citric Acid Cycle

Berg, Tymoczko & Stryer, 2012 Fig. 17.4

Glycolysis to the Citric Acid Cycle

Citric Acid Cycle

Pyruvate Dehydrogenase Complex

Preparation to enter the Citric Acid Cycle

Citric Acid Cycle

Pyruvate Dehydrogenase Reaction

Pyruvate + CoA + NAD+ acetyl CoA + CO2 + NADH

This is an irreversible reaction that links glycolysis and the citric acid cycle.

Citric Acid Cycle

Horton et al 2002, Table 12.1

Pyruvate Dehydrogenase Complex (E. Coli vs mammalian)

Citric Acid Cycle

Berg, Tymoczko & Stryer, 2012 Fig. 17.7

Pyruvate Dehydrogenase Complex Schematic

Citric Acid Cycle

/ Pyruvate Dehydrogenase

Horton et al, 2002 Fig. 12.3

E1

E2

E3

PDH Azobacter vinelandii

core

completeBerg, Tymoczko & Stryer, 2001 Fig. 17.3

Voet, Voet, & Pratt 2012 Fig. 17.4

Berg, Tymoczko & Stryer, 2012 Fig. 17.8

PDH Complex: Transacetylase (E2) Core

Citric Acid Cycle

/ Pyruvate Dehydrogenase

Berg, Tymoczko & Stryer, 2012 Chap 17 p.500

Two of the cofactors in the Pyruvate Dehydrogenase Complex

Citric Acid Cycle

Berg, Tymoczko & Stryer, 2012 Chap. 17 p. 500

PDH Complex’s Three Basic Reaction Types

Citric Acid Cycle

Lehninger 2000 Fig 16.6

Citric Acid Cycle/ /

Pyruvate Dehydrogenase

Oxidative Decarboyxlation of Pyruvate by the PDH Complex

Berg, Tymoczko & Stryer, 2012 Fig. 17.9

Pyruvate Dehydrogenase Complex Rx

Citric Acid Cycle

/ Pyruvate Dehydrogenase

Berg, Tymoczko & Stryer, 2002 Chap 17 p. 500

Formation of TPP Carbanion

Citric Acid Cycle

TPP is the prosthetic group of pyruvate dehydrogenase.

Pyruvate Dehydrogenase Complex: Mechanisms

Citric Acid Cycle

Berg, Tymoczko & Stryer, 2012 Fig. 17.6

Pyruvate Dehydrogenase Complex: Decarboxylation Reaction of E1

Citric Acid Cycle

/ Pyruvate Dehydrogenase

The charged TPP ring functions as an electron sink that acts to stabilize the transferred negative charge

Berg, Tymoczko & Stryer, 2002 Chap 17 p. 500

PDH Complex: Lipoamide Structure

Citric Acid Cycle

/ Pyruvate Dehydrogenase

Structures and Interconversion of Lipoamide & Dihydrolipoamide

Voet, Voet & Pratt 2013 Figure 17.7

Voet, Voet & Pratt 2013 Chap 17 p. 559

PDH Complex: Oxidation of the Hydroxyethyl Group and Transfer to Lipoamide

Citric Acid Cycle

/ Pyruvate Dehydrogenase

Catalyzed by pyruvate dehydrogenase component (E1).

Carbanion

PDH Complex: Formation of Acetyl CoA by Transfer of Acetyl Group from Acetyllipoamide

Citric Acid Cycle

/ Pyruvate Dehydrogenase

Catalyzed by dihydrolipoyl transacetylase (E2).

Voet, Voet & Pratt 2013 Chap 17 p. 559

PDH Complex: Regeneration of Oxidized Form of Lipoamide by Dihydrolipoyl Dehydrogenase

Citric Acid Cycle

/ Pyruvate Dehydrogenase

Voet, Voet & Pratt 2013 Chap 17 p. 559

Summary of Two-step Process above

Berg, Tymoczko & Stryer, 2012 Chap 17

The Citric Acid Cycle

Citric Acid Cycle

Leheninger 2000 Fig 16.7

Citric Acid Cycle Diagram: #11

Note that the acetyl group that enters the cycle does not give rise to the CO2 molecules given off in the decarboxylations in ONE TURN of the cycle.

Citrate Synthase Structure

OPEN CLOSED

Berg, Tymoczko & Stryer Figure 17.10

Oxaloacetate binding induces the two domains to move toward each other in an 18 degree arc

This forms a binding site for acetyl CoA.

Berg, Tymoczko & Stryer, 2012 Chap 17 p.504

Citric Acid Cycle: Condensation of Oxaloacetate & acetyl CoA

Citric Acid Cycle

Citrate synthaseCitrate synthase

G = -31.4 kJ mol-1Reaction 1

Berg, Tymoczko & Stryer, 2012 Fig. 17.11

Citric Acid Cycle: Synthesis of Citryl CoA by Citrate Synthase

Citric Acid Cycle

Leheninger 2000 Fig 16.7

Citric Acid Cycle Diagram: #2

2

The purpose of this reaction is to convert the citrate molecule to a secondary alcohol.Voet, Voet & Pratt, 2013 Chap 17 p.563

Citric Acid Cycle: Isomerization of Citrate by Aconitase

Citric Acid Cycle

Aconitase Aconitase

G = +8.4 kJ mol-1 G = -2.1 kJ mol-1

Reaction 2

Berg, Tymoczko & Stryer, 2012 Fig. 17.12

Citrate Binding to Aconitase’s Fe-S Complex

Citric Acid Cycle

Aconitase: Mechanism & Stereochemistry

Voet & Voet Biochemistry 1995 Fig. 19.3

Citric Acid Cycle

Leheninger 2000 Fig 16.7

Citric Acid Cycle Diagram: #3,4

3

4

Voet, Voet, & Pratt Fig. 17.11

Citric Acid Cycle: Oxidative Decarboxylation of Isocitrate by Isocitrate Dehydrogenase

Citric Acid Cycle

Isocitrate dehydrogenase

Isocitrate dehydrogenase

G = -8.4 kJ mol-1Reaction 3

Do not dissociate from enzyme

Berg, Tymoczko & Stryer, 2012 Chap 17 p.507

Citric Acid Cycle: Oxidative Decarboxylation of -ketoglutarate

Citric Acid Cycle

-ketoglutarate dehydrogenase complex

G = -30.1 kJ mol-1Reaction 4

Leheninger 2000 Fig 16.7

Citric Acid Cycle Diagram: #5

5

Berg, Tymoczko & Stryer, 2012 Chap 17 p.508

Citric Acid Cycle: Succinyl CoA Synthetase Reaction

Citric Acid Cycle

Succinyl CoA Synthetase

G = -3.3 kJ mol-1Reaction 5

Berg, Tymoczko & Stryer, 2012 Fig. 17.13

Citric Acid Cycle: Succinyl CoA Synthetase Rx Mechanism

Citric Acid Cycle

Rx’s of Succinyl-CoA Synthetase

Voet & Voet , & Pratt 2013 Fig. 17.12Citric Acid Cycle

Leheninger 2000 Fig 16.7

Citric Acid Cycle Diagram: #6,7

6

7

Voet, Voet & Pratt 2013 Chap 17 p. 567

Citric Acid Cycle: Succinate Dehydrogenase Rx

Citric Acid Cycle

Succinate dehydrogenase

G = 0 kJ mol-1Reaction 6

FAD vs NAD+ Reduction

In general:

FAD functions biochemically to oxidize alkanes to alkenes. The oxidation of an alkane, e.g. succinate, to an alkene (fumarate) is sufficiently exergonic to reduce FAD to FADH2 but not to reduce NAD+.

NAD+ oxidizes alcohols to aldehydes or ketones. Alcohol oxidation can reduce NAD+ to NADH

Voet, Voet & Pratt 2013 p. 567; Voet & Voet 1996 p555Citric Acid Cycle

Voet, Voet, & Pratt 2012 Chap. . 17 p. 567

Citric Acid Cycle: Hydration of Fumarate to Malate by Fumarase

Citric Acid Cycle

Fumarase

G = -3.8 kJ mol-1Reaction 7

Fumarase

Berg, Tymoczko & Stryer, 2012 Chap. 17 p.510

Citric Acid Cycle

Fumarate/Malate Stereochemistry

Voet, Voet & Pratt 2006 Figure page 531

Voet, Voet, & Pratt 2013 Chap 17 p. 567

Citric Acid Cycle: Oxidation of Malate to Oxaloacetate By Malate Dehydrogenase

Citric Acid Cycle

Malate dehydrogenase

G = +29.7 kJ mol-1Reaction 8

Citric Acid Cycle Stoichiometry

Citric Acid Cycle

Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O

2 CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA

Berg, Tymoczko & Stryer, 2012 Fig. 17.15

Citric Acid (Krebs) Cycle

Citric Acid Cycle

Berg, Tymoczko & Stryer, 2012 Table 17.2

Citric Acid (Krebs) Cycle Reactions

Citric Acid Cycle

Citric Acid Cycle

Stoichiometry of ATP Formation Table

Regulation of Entry Into and Metabolism Throughthe Citric Acid Cycle

Pathway from Glucose to Acetyl CoA

Citric Acid Cycle Horton et la, , 2012 Fig. 13.11 Voet, Voet & Pratt 2013 Chap 17 page 569

Berg, Tymoczko & Stryer, 2002 Fig. 17.17

Regulation of the Pyruvate Dehydrogenase Complex

Citric Acid Cycle

Berg, Tymoczko & Stryer, 2012 Fig. 17.18a

Berg, Tymoczko & Stryer, 2012 Fig. 17.18b

Berg, Tymoczko & Stryer, 2012 Fig. 17.17

Control of Metabolic Flux in the CycleKey Factors:

• Substrate Availability

• Inhibition by accumulating products

• Allosteric feedback inhibition of enzymes that catalyze the cycle’s early reactions.

Lehninger 2000, p 587

Citric Acid Cycle

Enzyme Control Points:

Citrate synthase (Bacteria)

Isocitrate dehydrogenase

-ketoglutarate

Berg, Tymoczko & Stryer, 2012 Fig. 17.19

Control of the Citric Acid Cycle

Citric Acid Cycle Voet, Voet, & Pratt 2013 Fig. 17.16

The Citric Acid Cycle and Biosynthetic Precursors

Citric Acid Cycle

Citric Acid Cycle

Anaplerotic Reactions Table(most common anaplerotic reactions)

Serve to replenish the citric acid cycle intermediates that are removed as biosynthetic precursors

Degradative Pathways Generating Cycle Intermediates

• Oxidation of odd chain fatty acids lead to the production of succinyl-CoA

• Breakdown of the amino acids leucine, methionine and valine also lead to succinyl CoA production

• Transamination and Deamination of amino acids leads to the production of -ketoglutarate and oxaloacetate.

Berg, Tymoczko & Stryer, 2012 Fig. 17.20

Citric Acid Cycle: Roles in Biosynthesis

Citric Acid Cycle

Citric Acid Cycle

The Citric Acid Cycle in Anabolism: Diagram

End of Lectures