tca cycle; krebs cycle; citric acid...

Tricarboxylic Acid CycleTricarboxylic Acid CycleTCA Cycle; Krebs Cycle;

Citric Acid CycleTCA Cycle; Krebs Cycle;

Citric Acid Cycle

The Bridging Step: Pyruvate D’haseThe Bridging Step: Pyruvate D’hase

pyruvatepyruvate

H3C - C - CH

3C - C - C

OO

O-O-

OO NAD+ NAD+

NADH NADH

CoASHCoASH

CO2CO2

H3C - C - S - H

3C - C - S -

OO

CoACoA

acetyl CoAacetyl CoA

Pyruvate D’hase ComplexPyruvate D’hase Complex

• Multienzyme complex (E. coli enzyme has 60 subunits)

• Multienzyme complex (E. coli enzyme has 60 subunits)

• Three activities: pyruvate d’hase (E1); dihydrolipoyl transacetylase (E2); dihydrolipoyl d’hase (E3)

• Three activities: pyruvate d’hase (E1); dihydrolipoyl transacetylase (E2); dihydrolipoyl d’hase (E3)

• Prime example of metabolite channeling (substrates acted upon immediately on enzyme surface - no diffusion into cytosol)

• Prime example of metabolite channeling (substrates acted upon immediately on enzyme surface - no diffusion into cytosol)

1) Pyruvate d’hase : loss of CO2 from pyruvate with transfer of the remaining two-carbon unit as a hydroxyethyl group (thiamine pyrophosphate (TPP) used as a cofactor)

1) Pyruvate d’hase : loss of CO2 from pyruvate with transfer of the remaining two-carbon unit as a hydroxyethyl group (thiamine pyrophosphate (TPP) used as a cofactor)2) Dihydrolipoyl transacetylase : hydroxyethyl group transferred to lipoic acid and oxizided to a carboxylic acid (lipoic acid cofactor converted to acetyl dihydrolipoamide); acetyl group transferred to CoA [arsenic binds lipoamide]

2) Dihydrolipoyl transacetylase : hydroxyethyl group transferred to lipoic acid and oxizided to a carboxylic acid (lipoic acid cofactor converted to acetyl dihydrolipoamide); acetyl group transferred to CoA [arsenic binds lipoamide]

3. Dihydrolipoyl d’hase : lipoic acid regenerated using NAD+ and FAD; NADH is produced3. Dihydrolipoyl d’hase : lipoic acid regenerated using NAD+ and FAD; NADH is produced

Thiamine (TPP); Riboflavin (FAD); Niacin (NAD+); Pantothenate (CoA); Lipoic AcidThiamine (TPP); Riboflavin (FAD); Niacin (NAD+); Pantothenate (CoA); Lipoic Acid

E1E1 E2E2 E3E3

TPPTPP

CH3-C-HCH3-C-H

OHOH

SS

SS

FADFAD

E1E1 E2E2 E3E3

TPPTPPSS

SS

FADFAD

CH3-C-CH3-C-

OO

Acetyl CoAAcetyl CoA

E1E1 E2E2 E3E3

TPPTPP HSHS

HSHS

FADFAD

E1E1 E2E2 E3E3

TPPTPPSS

SS

FADH2FADH2

NAD+NAD+ NADHNADH

E1E1 E2E2 E3E3

TPPTPPSS

SS

FADFAD

Regulation of Pyruvate D’haseRegulation of Pyruvate D’hase

Acetyl CoA and NADH allosterically inhibit (product inhibition)

Acetyl CoA and NADH allosterically inhibit (product inhibition)

Mammalian pyr. d’hase is phosphorylated and inactivated by a pyr. d’hase kinase.

This kinase itself is activated allosterically by NADH and acetyl CoA. This effect is

reversed by pyr. d’hase phosphatase, which removed the phosphate and reactivates the

enzyme.

Mammalian pyr. d’hase is phosphorylated and inactivated by a pyr. d’hase kinase.

This kinase itself is activated allosterically by NADH and acetyl CoA. This effect is

reversed by pyr. d’hase phosphatase, which removed the phosphate and reactivates the

enzyme.

AMP activates and GTP inhibits pyruvate dehydrogenase. This commits pyruvate to

energy production.

AMP activates and GTP inhibits pyruvate dehydrogenase. This commits pyruvate to

energy production.

CH2CH2

OO

OO OHOH

OHOH

PO32-PO32-

OO

OO OO

CH2CH2O-P-O-P-OO-P-O-P-O

O-O-O-O-C-CH2-CH2-NH-C-CH-C-CH2-C-CH2-CH2-NH-C-CH-C-CH2-

AACH3CH3

CH3CH3

NHNH

SHSH

CH2CH2

Coenzyme ACoenzyme A 3’-5’-ADP3’-5’-ADP

Pantothenic acidPantothenic acid

β-Mercaptoethylamineβ-Mercaptoethylamine

1. Citrate Synthase1. Citrate Synthase

COO-COO-

COO-COO-

H2CH

2CCC O O

oxaloacetate (OAA)

oxaloacetate (OAA)

CoASHCoASH

acetyl CoAacetyl CoA

COO-COO-

COO-COO-

H2CH

2CCC HO HO

H2CH

2C

COO-COO-

COO-COO-

citratecitrate

Regulation of Citrate Sythase:Regulation of Citrate Sythase:

Reaction has a large negative DG = -53.9 kJ/mol

Reaction has a large negative DG = -53.9 kJ/mol

NADH and Succinyl CoA are allosteric inhibitors

NADH and Succinyl CoA are allosteric inhibitors

2. Aconitase2. Aconitase

COO-COO-

COO-COO-

H2CH

2CCC HO HO

H2CH

2C

COO-COO-

COO-COO-

citratecitrate

COO-COO-

OHOHCCCC H H

H2CH

2C

COO-COO-

COO-COO-

isocitrateisocitrateCOO-COO-

H H

3. Isocitrate D’hase3. Isocitrate D’hase

COO-COO-

OHOHCCCC H H

H2CH

2C

COO-COO-

COO-COO-

isocitrateisocitrateCOO-COO-

H H

CO2

CO2

NADH NADH NAD+ NAD+

α-ketoglutarate (αKg)

α-ketoglutarate (αKg)

CCCC H

2 H

2

H2CH

2C COO-COO-

COO-COO-OO

Regulation of Isocitrate D’haseRegulation of Isocitrate D’haseMammalian enzyme: NADH and ATP are allosteric inhibitors; ADP and NAD+ are

allosteric activators

Mammalian enzyme: NADH and ATP are allosteric inhibitors; ADP and NAD+ are

allosteric activators

E. Coli enzyme: phosphorylation of the enzyme by a specific protein kinase

abolishes activity; removal of the phosphate by a phosphatase restores activity

E. Coli enzyme: phosphorylation of the enzyme by a specific protein kinase

abolishes activity; removal of the phosphate by a phosphatase restores activity

4. a-Ketoglutarate D’hase4. a-Ketoglutarate D’hase

α-ketoglutarate (αKg)

α-ketoglutarate (αKg)

CCCC H

2 H

2

H2CH

2C COO-COO-

OO COO-COO- CO2CO2

CoASHCoASH NAD+NAD+

NADHNADH

Succinyl-CoASuccinyl-CoA

CCCC H

2 H

2

H2CH

2C COO-COO-

OOSCoASCoA

Reaction mechanism identical to that of pyruvate d’hase: same cofactors utilized (succinyl group transferred)

Reaction mechanism identical to that of pyruvate d’hase: same cofactors utilized (succinyl group transferred)

5. Succinyl CoA Synthetase5. Succinyl CoA Synthetase

Succinyl-CoASuccinyl-CoA

CCCC H

2 H

2

H2CH

2C COO-COO-

OOSCoASCoA

CoASHCoASH

GDP, PiGDP, PiGTPGTP

SuccinateSuccinate

CCCC H

2 H

2

H2CH

2C COO-COO-

OOO-O-

6. Succinate D’hase6. Succinate D’hase

SuccinateSuccinate

CC H2

H2

H2CH

2C COO-COO-

COO-COO-

FADFADFADH2FADH2

FumarateFumarate

-OOC-OOCCCCC

COO-COO-HH

HH

Regulation of Succinate D’haseRegulation of Succinate D’hase

Enzyme is a large multisubunit enzyme with muliple cofactors like pyruvate d’hase.

Enzyme is a large multisubunit enzyme with muliple cofactors like pyruvate d’hase.

Enzyme transfers electrons from the substrate succinate to ubiquinone (Q)Enzyme transfers electrons from the

substrate succinate to ubiquinone (Q)

Malonate (analogue of succinate) is a competitive inhibitor and blocks the cycle at

this step; αkg, citrate, succinate accumulate in its presence

Malonate (analogue of succinate) is a competitive inhibitor and blocks the cycle at

this step; αkg, citrate, succinate accumulate in its presence

7. Fumarase7. Fumarase

FumarateFumarate

-OOC-OOCCCCC

COO-COO-HH

HH

H2OH2O

MalateMalate

-OOC-OOCCH

2CH

2

CCCOO-COO-

HOHO HH

8. Malate D’hase8. Malate D’hase

MalateMalate

-OOC-OOCCH

2CH

2

CCCOO-COO-

HOHO HH NAD+NAD+NADHNADH

OAAOAA

-OOC-OOCCH

2CH

2

CCCOO-COO-

OO

Overall Equation for TCA:Overall Equation for TCA:Acetyl CoA + 3NAD+ + Q(FAD) + GDP + Pi + 2H

2O Acetyl CoA + 3NAD+ + Q(FAD) + GDP + Pi + 2H

2O

CoASH + 3NADH + QH2 (FADH

2) + GTP + 2CO

2 + 2H+CoASH + 3NADH + QH

2 (FADH

2) + GTP + 2CO

2 + 2H+

*No net degradation of intermediates in TCA

Cycle - they are reformed with each full turn of the cycle.

*No net degradation of intermediates in TCA Cycle - they are reformed with each full turn of the cycle.

*NADH and QH2 are oxidized by the respiratory electron transport chain. 3ATP per NADH and 2ATP per QH2.*NADH and QH2 are oxidized by the respiratory electron transport chain. 3ATP per NADH and 2ATP per QH2.

ReactionReactionEnergy Yielding ProductEnergy Yielding Product ATP’sATP’s

Isocitrate D’hase NADH 3Isocitrate D’hase NADH 3

α−Kg D’hase NADH 3α−Kg D’hase NADH 3

Succinyl CoA Synthetase GTP (ATP) 1Succinyl CoA Synthetase GTP (ATP) 1

Succinate D’hase QH2 2Succinate D’hase QH2 2

Malate D’hase NADH 3Malate D’hase NADH 3

1212One Round of TCAOne Round of TCA

Amount of ATP formed per 1 Glucose:Amount of ATP formed per 1 Glucose:

ATP’sATP’s

Glycolysis Glycolysis 88

Pyruvate D’hasePyruvate D’hase 66

TCATCA 2424

3838

The Glyoxylate CycleThe Glyoxylate Cycle

A “shunt” within the TCA cycleA “shunt” within the TCA cycle

• Biosynthetic route that leads to formation of glucose from acetyl CoA

• Biosynthetic route that leads to formation of glucose from acetyl CoA

• Occurs in plants, bacteria and yeast• Occurs in plants, bacteria and yeast

Isocitrate is cleaved by isocitrate lyase to form succinate and glyoxylate:Isocitrate is cleaved by isocitrate lyase to form succinate and glyoxylate:

H H COO-COO-

OHOHCCCC

H2CH

2C

COO-COO-

COO-COO-

isocitrateisocitrateCOO-COO-

H HSuccinateSuccinate

CC H2

H2

H2CH

2C COO-COO-

COO-COO-

CCOO HH

COO-COO-

GlyoxylateGlyoxylate

Glyoxylate condenses with acetyl CoA to form malate:

Glyoxylate condenses with acetyl CoA to form malate:

CCOO HH

COO-COO-

GlyoxylateGlyoxylate

++CH

3CH

3

C=OC=OS-CoAS-CoA

Acetyl CoAAcetyl CoA

MalateSynthaseMalateSynthase

MalateMalate

COO-COO-

CH2

CH2

CCCOO-COO-

HOHO HH

NO CARBON ATOMS LOST AS CO2! THUS A NET SYNTHESIS OF MALATE IS ACHEIVED.

NO CARBON ATOMS LOST AS CO2! THUS A NET SYNTHESIS OF MALATE IS ACHEIVED.

OAAOAA

CitrateCitrate

IsocitrateIsocitrate

αKgαKg

Acetyl CoAAcetyl CoA

Succinyl CoASuccinyl CoA

SuccinateSuccinate

FumarateFumarate

MalateMalate

GlyoxylateGlyoxylate

GlucoseGlucoseAcetyl CoAAcetyl CoA

CoASHCoASH

Glyoxylate Cycle requires transfer of metabolites between the mitochondrion, cytosol and a special organelle, the glyoxysome.

Glyoxylate Cycle requires transfer of metabolites between the mitochondrion, cytosol and a special organelle, the glyoxysome.

Glyoxysome: Isocitrate cleaved to succinate and glyoxylate. Glyoxylate condenses with acetyl CoA to form malate. Succinate goes to mitochondrion; malate to cytosol.

Glyoxysome: Isocitrate cleaved to succinate and glyoxylate. Glyoxylate condenses with acetyl CoA to form malate. Succinate goes to mitochondrion; malate to cytosol.

Mitochondrion: Succinate enters the TCA cycle.Mitochondrion: Succinate enters the TCA cycle.

Cytosol: Malate converted to OAA; OAA to glucose by the gluconeogenesis pathway.Cytosol: Malate converted to OAA; OAA to glucose by the gluconeogenesis pathway.