Citric Acid Cycle

CHEM 111

Additional Energy Can Be Extracted

From Pyruvate after Glycolysis

Complete Oxidation of Glucose:

C 6 H 12 O 6 + 6 O2  6 CO 2 + 6 H 2 O

ΔGo' = -2,840 kJ/mol

Glycolysis: ΔG o’ = -85 kJ/mol

A lot of energy remains in Pyruvate.

Under aerobic conditions, this

energy is extracted through the

Citric Acid Cycle

Stage 1: Acetyl-CoA Production

Oxidative decarboxylation of pyruvate: carboxyl group of pyruvate leaves as CO2

• Pyruvate diffuses into mitochondria through

large pores in outer membrane

• Then transported across inner membrane via

H+-coupled pyruvate specific symporter

• Mitochondrial Pyruvate Carrier (MPC)

• Pyruvate that enters mitochondrial matrix is

converted to Acetyl-CoA for further oxidation

or to serve as precursors for other

macromolecules

Overall PDH Reaction

Enzyme 1 – Pyruvate Dehydrogenase

• Step 1: Decarboxylation of pyruvate,

forming CO 2 and hydroxyethyl TPP.

• Step 2: Two electrons and the acetyl

group are transferred from TPP to the

oxidized form of the lipoyllysine in E2.

Enzyme 2 – Dihydrolipoyl Transacetylase

• Step 3: Formation of acetyl-CoA, and

reduction of lipoyllysine.

Enzyme 3 – Dihydrolipoyl Dehydrogenase

• Step 4: Reoxidation of lipoyllysine (FAD

accepts both electrons, H).

• Step 5: Regeneration of oxidized FAD

(NAD+ is reduced).

Enzyme 1 – Pyruvate Dehydrogenase

• Step 1: Decarboxylation of pyruvate,

forming CO 2 and hydroxyethyl TPP.

• Step 2: Two electrons and the acetyl

group are transferred from TPP to the

oxidized form of the lipoyllysine in E2

Thiamine Pyrophosphate (TPP) in C-C Bond Cleavage Reactions

Enzyme 1 – Pyruvate Dehydrogenase

Step 1: Decarboxylation of pyruvate,

forming CO 2 and hydroxyethyl TPP.

• Step 2: Two electrons and the acetyl

group are transferred from TPP to the

oxidized form of the lipoyllysine in E2.

Enzyme 2 – Dihydrolipoyl Transacetylase

Step 3: Formation of acetyl-CoA, and

reduction of lipoyllysine.

Enzyme 3 – Dihydrolipoyl Dehydrogenase

• Step 4: Reoxidation of lipoyllysine (FAD

accepts both electrons, H).

• Step 5: Regeneration of oxidized FAD

(NAD+ is reduced)

Pyruvate Dehydrogenase (PDH) Complex

PDH contains multiple copies of three enzymes (E1, E2, and E3)

Low-resolution CryoEM structure of PDH complex

High-resolution crystal

structures of E1 (left), E2

(right), and E3 (below)

Stage 2: Acetyl-CoA Oxidation

1 molecule of Acetyl-CoA  3 NADH, 1 FADH2 , and 1 ATP (or GTP)

The pathway that performs this chemistry

is called the Citric Acid Cycle, also known

as the Tricarboxylic Acid (TCA) Cycle

Overview of the Citric Acid Cycle

Chemical Logic of the Citric Acid Cycle

After the PDH reaction, we are basically left with acetate in the form of acetyl-CoA.

• There is still chemical energy that can be extracted via oxidation.

• Acetate/acetyl-CoA do not have functional groups positioned to facilitate

oxidation chemistry.

• Every reaction of the Citric Acid Cycle is either:

1. An oxidation (with or without accompanying decarboxylation).

2. Positioning functional groups to facilitate oxidation or oxidative decarboxylation

Decarboxylation of a β-keto acid:

Details of the citric acid cycle

Follow the Carbons:

•Carbons from acetate (coming from glucose) are

in red. None of them are lost in one turn of the

cycle.

•All CO2 generated during the citric acid cycle is

produced before succinyl-CoA is made. Both CO2

molecules lost were present on the oxaloacetate

used to begin the cycle.

•Succinate is symmetrical, so we can’t keep track

anymore.

Step 1: Addition to Oxaloacetate – Formation of Citrate

Acetyl-CoA enters TCA

cycle

• Addition is an aldol

condensation catalyzed by

citrate synthase

• Forms pro-chiral product –

citrate

Importance of Prochirality

Step 2: Isomerization – Formation of Isocitrate via cis-

Aconitate

Prochiral tertiary alcohol is converted

to a chiral secondary alcohol

• Isomerization occurs in two steps

both catalyzed by the same enzyme

• Dehydration takes place on the pro-R

arm of citrate

Iron-sulfur center of aconitase

• 3 Cys bind 3 Fe atoms

• 4th Fe is bound to one of the carboxyl groups of

Citrate and interacts noncovalently with the hydroxyl

group

• Basic residue helps position Citrate in active site

Step 3: Oxidative Decarboxylation – Oxidation of Isocitrate to

α-Ketoglutarate and CO

Oxidation and decarboxyation of Isocitrate to α-Ketoglutarate

Step 4: Oxidative Decarboxylation – Oxidation of α-Ketoglutarate to Succinyl-CoA and CO 2

Reaction Catalyzed by α-Ketoglutarate Dehydrogenase Complex:

Thiamine Pyrophosphate (TPP) in

C-C Bond Cleavage Reactions

Step 4: Oxidative Decarboxylation – Oxidation of α-

Ketoglutarate to Succinyl-CoA and CO 2

Step 5: Acyl-CoA cleavage – Conversion of Succinyl-CoA to

Succinate

Enzyme does not use metal cation cofactors,How does this enzyme stabilize the highly

charged His-Pi intermediate?

Utilizes dipole moment of two α-helices to stabilize the charged His-Pi intermediate

The Last Three Reactions Regenerate Oxaloacetate

• First, a C-C single bond in Succinate

is oxidized to a double bond, giving

Fumarate. Electrons and protons are

transferred to FAD, reducing it to

FADH 2.

• Next, the double bond in Fumarate

gets hydrated, yielding Malate.

• Finally, Malate is oxidized to

Oxaloacetate. NAD+ is reduced to

NADH.

Step 6: Dehydrogenation – Oxidation of Succinate to Fumarate

Stereospecific reaction, removing pro-S

hydrogen from one carbon and pro-R

hydrogen from the other

Step 7: Hydration – Hydration of Fumarate to Malate

Step 8: Oxidation – Oxidation of Malate to Oxaloacetate

Overview of the Citric Acid Cycle

• Net yields for every one cycle:

• 4 NADH

• 1 from Pyruvate to Acetyl-

CoA

• 3 from TCA cycle

• 1 FADH 2

• 2 CO 2

• 1 GTP (ATP)

Acetyl − CoA + 3NAD+ + FAD + GDP + Pi + 2H2O → 2CO2 + CoA − SH + 3NADH + 3H+ + FADH2 + GTP

Energy of Oxidations in Cycle is Efficiently Conserved

TCA Cycle is the Hub of Intermediary Metabolism

Amino Acids can Enter the CAC

The CAC in Anabolism