Citric Acid Cycle

Figure 17-2

Citric Acid Cycle

Summary of Citric Acid Cycle

Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi

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

Reactions of the Citric Acid Cycle

Citrate Synthase(citrate condensing enzyme)

Acetyl- SCoA Oxaloacetate

H3C C

O

S CoA

C

H2C

COOH

COOH

O+

H2C

C

H2C

HO

COOH

COOH

COOH

Citrate

CoA–SH

∆Go’ = –31.5 kJ/mol

Figure 17-10 part 1

Mechanism of Citrate Synthase

(Formation of Acetyl-SCoA Enolate)

Figure 17-10 part 2

Mechanism of Citrate Synthase

(Acetyl-CoA Attack on Oxaloacetate)

Figure 17-10 part 2

Mechanism of Citrate Synthase

(Hydrolysis of Citryl-SCoA)

Regulation of Citrate Synthase

• Pacemaker Enzyme (rate-limiting step)

• Rate depends on availability of substrates

– Acetyl-SCoA

– Oxaloacetate

Aconitase

Stereospecific

Addition

Cis- aconitate(~3%)

I socitrate(~6%)

H2C

C

H2C

HO

COOH

COOH

COOH

Citrate(~91%)

H2C

C

HC

COOH

COOH

COOH

H2C

HC

CH

COOH

COOH

COOH

HO

H2OH2O

∆Go’ = ~0

Iron-Sulfur Complex(4Fe-4S]

Thought to coordinate citrate –OH to facilitate elimination

Page 325

Stereospecificity of Aconitase Reaction

Prochiral Substrate Chiral Product

Figure 11-2

Stereospecificity in Substrate Binding

NAD+–DependentIsocitrate Dehydrogenase

I socitrate

H2C

HC

CH

COOH

COOH

COOH

HO

NAD+ NADH + H+

- ketoglutarate

H2C

CH2

C

COOH

COOHO

+ CO2

Mn2+ or Mg2+

Oxidative Decarboxylation

NOTE: CO2 from oxaloacetate

∆Go’ = -20.9 kJ/mol

Figure 17-11 part 1

Mechanism of Isocitrate Dehydrogenase

(Oxidation of Isocitrate)

Figure 17-11 part 2

Mechanism of Isocitrate Dehydrogenase

(Decarboxylation of Oxalosuccinate)

Mn2+ polarizes C=O

Figure 17-11 part 2

Mechanism of Isocitrate Dehydrogenase(Formation of -Ketoglutarate)

Regulation of Isocitrate Dehydrogenase

• Pulls aconitase reaction

• Regulation (allosteric enzyme)

– Positive Effector: ADP (energy charge)

– Negative Effector: ATP (energy charge)

• Accumulation of Citrate: inhibits Phosphofructokinase

Accumulation of Citrate

CO2

Isocitrate dehydrogenase

CO2

Isocitrate dehydrogenase

-Ketoglutarate Dehydrogenase

NAD+ NADH + H+

- ketoglutarate

H2C

CH2

C

COOH

COOHO

+ CO2+ CoASH

Succinyl- CoA

H2C

CH2

C

COOH

SCoAO

Oxidative Decarboxylation

Mechanism similar to PDH

CO2 from oxaloacetate

High energy thioester

∆Go’ = -33.5 kJ/mol

-Ketoglutarate Dehydrogenase

(Multienzyme Complex)

• E1: -Ketoglutarate Dehydrogenase or -Ketoglutarate Decarboxylase

• E2: Dihydrolipoyl Transsuccinylase

• E3: Dihydrolipoyl Dehydrogenase (same as E3 in PDH)

Regulation of -Ketoglutarate Dehydrogenase

• Inhibitors

– NADH

– Succinyl-SCoA

• Activator: Ca2+

Origin of C-atoms in CO2

H2C

C

H2C

HO

COOH

COOH

COOH

Citrate I socitrate

H2C

HC

CH

COOH

COOH

COOH

HO

- ketoglutarate

H2C

CH2

C

COOH

COOHO

Succinyl- CoA

H2C

CH2

C

COOH

SCoAO

Both CO2 carbon atoms derived from oxaloacetate

Succinyl-CoA Synthetase(Succinyl Thiokinase)

GDP + Pi GTP

+ CoASH

Succinyl- CoA

H2C

CH2

C

COOH

SCoAO

H2C

H2C

COOH

COOH

Succinate

High Energy Thioester —> Phosphoanhydride Bond

Plants and Bacteria: ADP + Pi —> ATP

Randomizationn of labeled C atoms

∆Go’ = ~0

Thermodynamics(Succinyl-SCoA Synthetase)

Succinyl-SCoA+ H2O Succinate + CoA

GDP + Pi GTP + H2O

Succinyl-SCoA + GDP + Pi

² Go' = –32.6 kJ / mol

² Go' = +30.5 kJ / mol

² Go' = –2.1 kJ /molSuccinate + GTP + CoA

Page 581

Evidence for Phosphoryl-enzyme Intermediate

(Isotope Exchange)

Absence of Succinyl-SCoA

Figure 17-12 part 1

Mechanism of Succinyl-CoA Synthetase

(Formation of High Energy Succinyl-P)

Figure 17-12 part 2

Mechanism of Succinyl-CoA Synthetase

(Formation of Phosphoryl-Histidine)

Figure 17-12 part 3

Mechanism of Succinyl-CoA Synthetase(Phosphoryl Group Transfer)

Substrate-level phosphorylation

Nucleoside Diphosphate Kinase

(Phosphoryl Group Transfer)

GTP + ADP ——> GDP + ATP

∆Go’ = ~0

Succinate Dehydrogenase

Randomization of C-atom Labeling

Membrane-Bound Enzyme

H2C

H2C

COOH

COOH

Succinate

CH

HC COOH

HOOC

Fumarate

FAD FADH2

∆Go’ = ~0

Figure 17-13

Covalent Attachment of FAD

FAD used for Alkane Alkene

• Reduction Potential– Affinity for electrons; Higher E, Higher Affinity

– Electrons transferred from lower to higher EEh

o’ = Go’/nF = -(RT/nF)ln (Keq)

FAD/FADH2

Succinate/Fumarate

NAD+/NADH

Isocitrate/α-Ketoglutarate

Reduction Potential

Fumarase

H2O

CH

HC COOH

HOOC

Fumarate

HC

H2C COOH

HO COOH

Malate

∆Go’ = ~0

Page 583

Mechanism of Fumarase

Malate Dehydrogenase

NAD+ NADH + H+

HC

H2C COOH

HO COOH

Malate

C

H2C COOH

O COOH

Oxaloacetate

∆Go’ = +29.7 kJ/mol

Low [Oxaloacetate]

Thermodynamics

Malate + NAD+ Oxaloacetate + NADH + H+

Acetyl-SCoA + Oxaloacetate Citrate + CoA

Malate + NAD+

+ Acetyl-SCoA

² Go' = +29.7 kJ / mol

² Go' = –31.5 kJ / mol

² Go' = –1.8 kJ /molNADH + H+ +

Citrate + CoA

Figure 17-14

Products of the Citric Acid Cycle

Page 584

ATP Production from Products

of the Central metabolic Pathway

= 32 ATP

NADH 2.5 ATPFADH2 1.5 ATP

Amphibolic Nature of Citric

Acid Cycle

Carbons of Glucose:1st cycle

1 2 36 5 4

3, 4

2,51,6

2,51,61,62,5

2,51,61,62,5

Carbons of Glucose:2nd cycle:

Carbons 2,5:After 1½ turns:all radioactivity is CO2

Carbons of Glucose:2nd cycle:

Carbons 1,6:After 2 turns:¼ radioactivity in each carbon of OAA

Carbons of Glucose:3rd cycle:

Carbons 1,6:After 3 turns:½ radioactivity is CO2

Each turn after willlose ½ remainingradioactivity

Carbon Tracing from Glucose

• Glucose radiolabeled at specific Carbons– Can determine fate of individual carbons

• Carbons 1,6– 1st cycle: 1, 4 of oxaloacetate– Starting at 3rd cycle ½ radioactivity CO2/cycle

• Carbons 2,5– 1st cycle: 2, 3 of oxaloacetate

– 2nd cycle: all eliminated as CO2

• Carbons 3,4– All eliminated at CO2 during Pyruvate Acetyl-CoA

You are following the metabolism of pyruvate in which the methyl-carbon is radioactive: *CH3COCOOH.

-assuming all the pyruvate enters the TCA cycle as Acetyl-CoA, indicate the labeling pattern and its distribution in oxaloacetate first formed by operation of the TCA cycle.

Generation of Citric Acid Cycle Intermediates

Pyruvate Carboxylase

Mitochondrial Matrix

Pyruvate Carboxylase

Animals and Some Bacteria

ATP

HCO3–

(CO2)H3C C COOH

O

COOH

CH2

CO COOH

Oxaloacetate

ADP + Pi

+

PyruvatePyruvate

Carboxylase

Biotin Cofactor(CO2 Carrier)

NHC

HN

H2CS

CH

O

(CH2)4 C NH

O

(CH2)4 CH

C

NH

O

Biotin

Lysine

Reaction Mechanism I(Dehydration/Activation of HCO3

–)

NHC

HN

H2CS

CH

O

(CH2)4 C NH

O

(CH2)4 Enzyme

O P O

O

O–

P

O

O–

O–AMP –O COH

O

HCO3–

ATP

CO

–ONH

CN

H2CS

CH

O

(CH2)4 C NH

O

(CH2)4 Enzmye

Biotinyl-Enzyme

ADP + Pi

Carboxybiotinyl- Enzyme

Reaction Mechanism II(Transfer of CO2 to Pyruvate)

C C CH2–

OO

–O CO

–ONH

CN

H2CS

CH

O

(CH2)4 C NH

O

(CH2)4 EnzymeC C CH2

O–

O

–O

CO

O–C C CH2

OO

–O

Pyruvate EnolateCarboxybiotinyl-Enzyme

Oxaloacetate

Biotinyl- Enzyme

Fates of Oxaloacetate

Regulation!

COO–

C

CH3

O

ATP COO–

C

CH2

O

COO–

ADP + Pi

Pyruvate

+ HCO3–

Oxaloacetate

PyruvateCarboxylase

Gluconeogenesis

Citric AcidCycle

Regulation of Pyruvate Carboxylase

Allosteric ActivatorAcetyl-SCoA

Glyoxylate Cycle

Glyoxysome

Plants and Some Microorganisms

Citrate Synthase(citrate condensing enzyme)

Acetyl- SCoA Oxaloacetate

H3C C

O

S CoA

C

H2C

COOH

COOH

O+

H2C

C

H2C

HO

COOH

COOH

COOH

Citrate

CoA–SH

Aconitase

Cis- aconitate(~3%)

I socitrate(~6%)

H2C

C

H2C

HO

COOH

COOH

COOH

Citrate(~91%)

H2C

C

HC

COOH

COOH

COOH

H2C

HC

CH

COOH

COOH

COOH

HO

H2OH2O

Glyoxylate Cycle Enzymes

CHO

COOH

Glyoxylate

H2C

HC

CH

COOH

COOH

COOHHO

H3C C S–CoA

O

H2C

H2C

COOH

COOH

CoA-SH

CH

H2C

COOH

COOH

HO

CHO

COOH

Glyoxylate

+

SuccinateI socitrate

I socitrateLyase

Acetyl–SCoA Malate

+

MalateSynthase

Plants and Some Microorganisms

Malate Dehydrogenase

NAD+ NADH + H+

HC

H2C COOH

HO COOH

Malate

C

H2C COOH

O COOH

Oxaloacetate

Net Reaction of Glyoxylate Cycle

Net increase of one 4-carbon unit!

2 Acetyl-CoA 1 Oxaloacetate

Figure 17-18

Glyoxylate Cycle and the Glyoxysome

Regulation of the Citric Acid Cycle

Regulatory Mechanisms

• Availability of substrates– Acetyl-CoA– Oxaloacetate

– Oxygen (O2)

• Need for citric acid cycle intermediates as biosynthetic precursors

• Demand for ATP

Table 17-2

Free Energy Changes of Citric Acid Cycle Enzymes

Regulation of Pyruvate Dehydrogenase

• Product Inhibition (competitive)

– NADH

– Acetyl-SCoA

• Phosphorylation/Dephosphorylation

– PDH Kinase: inactivation

– PDH Phosphatase: reactivation

Figure 17-15

Covalent Modification and Regulation of PDH

Regulation of PDH Kinase(Inactivation)

• Activators– NADH– Acetyl-SCoA

• Inhibitors– Pyruvate– ADP– Ca2+ (high Mg2+)

– K+

Regulation of PDH Phosphatase(Reactivation)

• Activators– Mg2+

– Ca2+

Regulation of Citrate Synthase

• Pacemaker Enzyme (rate-limiting step)

• Rate depends on availability of substrates

– Acetyl-SCoA

– Oxaloacetate

Regulation of Isocitrate Dehydrogenase

• Pulls aconitase reaction

• Regulation (allosteric enzyme)

– Positive Effector: ADP (energy charge)

– Negative Effector: ATP (energy charge)

• Accumulation of Citrate: inhibits Phosphofructokinase

Regulation of -Ketoglutarate Dehydrogenase

• Inhibitors

– NADH

– Succinyl-SCoA

• Activator: Ca2+

Figure 17-16

Regulation of the Citric Acid Cycle