BC368: Biochemistry of the Cell IIBC368: Biochemistry of the Cell II

Citric Acid CycleChapter 16

March 12, 2015

3 stages of respiration3 stages of respiration

Production of acetyl-CoA (e.g., during glycolysis and the bridging reaction)

Oxidation of acetyl-CoA via the citric acid cycle

Electon transport and oxidative phosphorylation to produce lots of ATP

Fig 16-1

Glycolysis takes place in the cytosol

The citric acid cycle takes place in the mitochondrial matrix

Mitochondrial ArchitectureMitochondrial Architecture

The Bridging ReactionThe Bridging Reaction

H+ +

The Bridging ReactionThe Bridging Reaction

Fig 16-2

The Bridging ReactionThe Bridging Reaction

E1: orange

E2: green

E3: yellow

Fig 16-6

Pyruvate dehydrogenase complexPyruvate dehydrogenase complex

1. Decarboxylation 2. Oxidation3. Acetyl group to CoA 4. Restore enzyme

Fig 16-6

Fig 16-6

Pyruvate dehydrogenase complexPyruvate dehydrogenase complex

Fig 16-6

Step 1. Decarboxylation

Step 1: DecarboxylationStep 1: Decarboxylation

TPP is derived from vitamin B1

Common for decarboxylation reactions

Carries carbon groups transiently

Fig 14-15

Fig 16-6

Pyruvate dehydrogenase complexPyruvate dehydrogenase complex

Fig 16-6

Step 2. Oxidation, with reduction of E2

Step 2: OxidationStep 2: Oxidation

Hydroxyethyl group is oxidized to acetyl group, transferred to lipoamide of E2, which is reduced.

of interest here

Lipoic Acid “Swinging Arm”Lipoic Acid “Swinging Arm”

Swinging arm acyl group carrier

Transfers intermediates between different enzyme sites

The Marsh Test

Fig 16-6

Pyruvate dehydrogenase complexPyruvate dehydrogenase complex

Fig 16-6

Step 3. Transfer to CoA

Step 3: Transfer to CoAStep 3: Transfer to CoA

Acetyl group is transferred to coenzyme A by E2.

Coenzyme ACoenzyme A

Derived from Vitamin B5 (pantothenic acid)

“Activates” the acetyl group

Fig 16-3

Fig 16-6

Pyruvate dehydrogenase complexPyruvate dehydrogenase complex

Fig 16-6

Step 4. Restoring the enzyme

Fig 16-6

Step 4: Restoring the enzymeStep 4: Restoring the enzyme

FAD of E3 reoxidizes dihydrolipoamide.

NAD+ reoxidizes FADH2.

FAD/FADH2FAD/FADH2

Derived from Vitamin B2 (riboflavin)

1 or 2 electron acceptor

NAD+/NADHNAD+/NADH

Derived from Vitamin B3 (niacin)

2 electron acceptor

Fig 16-6

Pyruvate dehydrogenase complexPyruvate dehydrogenase complex

Fig 16-6

Coenzyme ACoenzyme A

Acetyl group is activated in two ways:

Carbonyl carbon is activated for attack by nucleophiles

Methyl carbon is more acidic

Fig 16-3

The Citric Acid CycleThe Citric Acid Cycle

Reaction 1: CondensationReaction 1: Condensation

Citrate synthase mechanism

Fig 16-9

1. deprotonation of methyl group of acetyl-CoA

2. enolate attacks carbonyl of OA, forming citroyl-CoA

Citrate synthase mechanism

Fig 16-9

3. hydrolysis of thioester releases citrate and CoA

Citrate synthase mechanism

Fig 16-9

Reaction 2: IsomerizationReaction 2: Isomerization

A symmetric molecule that acts asymmetric!

Chemically, these carbons are identical!

A symmetric molecule that acts asymmetric!

Chemically, these carbons are identical!

So both these products should be formed

A symmetric molecule that acts asymmetric!

Chemically, these carbons are identical!

So both these products should be formed

Prochiral molecules can act

chiral!

Reaction 3: Oxidative DecarboxylationReaction 3: Oxidative Decarboxylation

Reaction 4: Oxidative DecarboxylationReaction 4: Oxidative Decarboxylation

Reaction 5: Substrate-level phosphorylationReaction 5: Substrate-level phosphorylation

Succinyl-CoA synthetase reactionSuccinyl-CoA synthetase reaction

Hydrolysis of CoA-SH drives phosphorylation of succinate within the enzyme-substrate complex

Succinate transfers its phosphate group to the enzyme

Enzyme phosphorylates GDP

Reactions 6, 7, and 8Reactions 6, 7, and 8

Oxidation

Hydration

Oxidation

Summary of TCASummary of TCA

Fig 16-14

Regulation Regulation

Irreversible reactions are regulated

In general, energy charge is key:

AMP/NAD+ activateATP/NADH inhibit

Product inhibition

Fig 16-19

Anaplerotic ReactionsAnaplerotic Reactions

Fig 16-16

Anaplerotic ReactionsAnaplerotic Reactions

Example: pyruvate carboxylase, which uses a biotin (vitamin B7) cofactor to carry CO2

Daniel plans to enter the Mr. Colby contest and wants to get jacked. He has begun adding raw eggs to his diet and is up to a dozen a day. Unfortunately, he has been experiencing lactic acidosis during his weight training and hypoglycemia between meals. What’s up with Daniel?

Case Study

KD ≈ 10-15 M

Case Study

Pyruvate carboxylase

Carboxyl group of bicarbonate is “activated” by phosphorylation

Pyruvate carboxylase

“Activated” CO2 is passed to biotin cofactor with loss of Pi

Pyruvate carboxylase

CO2 is passed to second active site for rxn with pyruvate

CO2 is released for reaction with pyruvate to form OA.

Pyruvate carboxylase

Glyoxylate cycle

Fig 16-22

Plants and some microorganisms can convert acetyl-CoA to oxaloacetate for net gain of carbon and net synthesis of TCA intermediates

Intersection with TCA

Fig 16-24

Glyxoylate pathway runs simultaneously with TCA but in a different compartment.

Coordinated regulation

Fig 16-25

Isocitrate is a branch point; its fate depends on relative activities of isocitrate dehydrogenase (TCA) and isocitrate lyase (glyoxylate cycle).

Vania can’t believe that she feels so lousy. Even though it is St. Patrick’s Day weekend and she’s been up all night partying, she’s never felt this bad before. Her head is pounding, and she feels tired, weak, dizzy, and sick to her stomach. She would drink some water, but she lost her Nalgene bottle last week somewhere, and the walk to the dining hall is just way too far.

Case Study

1. What is wrong with Vania?2. What are the consequences of dehydration on metabolism?3. What are the metabolic breakdown products of ethanol?4. What role do these metabolic products play in the citric acid cycle? 5. What would you recommend to Vania?