Tricarboxylic Acid Cycle Tricarboxylic Acid Cycle TCA Cycle; Krebs Cycle; Citric Acid Cycle TCA Cycle; Krebs Cycle; Citric Acid Cycle
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.