TCA cycle

Where does the TCA cycle take place?

Mitochondrial matrix

AT this point, what stage is metabolism of glucose at?

Glycolysis done, PDH complex has produced Acetyl-CoA

• Acetyl-CoA also comes from other mechanisms of breakdown (B-oxidation, amino acid catabolism)

What is the overall putpose of TCA?

Complete oxidation of acetyl units to CO2 and capture energy

What is special about the TCA (what can product intermediates be used for)?

There is no one final product, goes around in cycle

Each intermediate can be siphoned off and used for other processes (catabolic and anabolic)

What type of procses is the TCA cycle?

cyclical pathway

Amphibolic pathway (catabolic and anabolic pathway)

• a NET catabolic pathway (more catabolism than anabolism)

How many steps is the TCA cycle? What are phases. Which steps are in which phases

8 steps of the TCA cycle

4 Phases of the cycle

1. Acetyl Entry (steps 1-2)

1. Oxidative Phase (steps 3-4)

2. Substrate-level phosphorylation (step 5)

3. Regeneration phase (step 6-8)

What happens in each phase of the TCA cycle?

Acetyl Entry (steps 1-2)

• Acetyl-CoA + Oxaloacetate → Citrate → Isocitrate

Oxidative Phase (steps 3-4)

• Two decarboxylations producing NADH + CO2

Substrate-level phosphorylation (step 5)

• Energy Captured as GTP/ATP (an ATP equivalent)

Regeneration Phase (steps 6-8)

• Rebuild Oxaloacetate + MORE NADH/FADH2

What is produced from ONE cycle of the TCA? What molecule runs the cycle? 

• 1 ATP

• 3 NADH

• 1 FADH2

• 2 CO2

ONE acetyl-CoA → ONE cycle of the TCA

For one molecule of Glucose, how many times does the TCA run? What is produced overall?

One glucose → two Acetyl-CoA → TWO cycles of the TCA

• 6 NADH

• 2 FADH2

• 2 GTP/ATP (depends on the tissue, 2 ATP equivalents)

• 4 CO2

List out all th Enzymes, reactants, and products for each phase of step of the TCA cycle. THIS IS ACETYL ENTRY PHASE

Acetyl Entry Phase

• Step 1

  • Reactant: Oxaloacetate + Acetyl-CoA

  • Enzyme: Citrate Synthase

  • Products: Citrate + CoA

• Step 2

  • Reactant: Citrate

  • Enzyme: Aconitase

  • Product: Isocitrate

List out all th Enzymes, reactants, and products for each phase of step of the TCA cycle. THIS IS OXIDATIVE PHASE

Oxidative Phase

• Step 3

  • Reactant: Isocitrate + NAD+

  • Enzyme: Isocitrate Dehydrogenase

  • alpha-ketoglutarate + NADH + CO2

• Step 4

  • Reactant: alpha-ketoglutarate + NAD+ + CoA

  • Enzyme: alpha-ketoglutarate dehydrogenase complex

  • Product: Succinyl CoA + NADH + CO2

List out all th Enzymes, reactants, and products for each phase of step of the TCA cycle. THIS IS SUBSTRATE-LEVEL PHOSPHORYLATION PHASE

Substrate Level Phosphorylation

• Step 5

  • Succinyl CoA + ADP + Pi

  • Enzyme: Succinyl CoA

  • Product: Succinate + ATP + CoA

List out all th Enzymes, reactants, and products for each phase of step of the TCA cycle. THIS IS REGENERATION PHASE

Regeneration Phase

• Step 6

  • Succinate + FAD

  • Enzyme: Succinate Dehydrogenase

  • Product: Fumarate + FADH2

• Step 7

  • Reactant: Fumarate + H2O

  • Enzyme: Fumarase (Fumarate Hydrolase)

  • Product: Malate

• Step 8

  • Reactant: Malate + NAD+

  • Enzyme: Malate Dehydrogenase

  • Product: Oxaloacetate + NADH

Which Steps produceH NADH and CO2? Which Step produces FADH2? What step produces GTP/ATP? Which steps produce CO2

NADH AND CO2

• Step 3.

Isocitrate + NAD+ —Isocitrate Dehydrogenase→ alpha-ketoglutarate + NADH + CO2

• Step 4

alpha-ketoglutarate + NAD+ + CoA —alpha-ketoglutarate dehydrogenase complex→ Succinyl CoA + NADH + CO2

JUST NADH

• Step 8

Malate + NAD+ —Malate Dehydrogenase→ NADH + H+

Which Step produces FADH2? What step produces GTP/ATP?

FADH2

• Step 6

Succinate + FAD —Succinate Dehydrogenase→ Fumarate + FADH2

GTP/ATP

• Step 5

Succinyl CoA + ADP + Pi —Succinyl CoA Synthetase→ Succinate + ATP + CoA

How much ATP does 1 molecule of NADH produce? What about FADH2?

NADH → 2.5 ATP

FADH2 → 1.5 ATP

Per glucose, how much ATP is produced from 1 molecule of Glucose (2 cycles beacuse 2 Acetyl-CoA produced from 2 pyruvate molecules)

6 NADH → 15 ATP (via ETC)

2 FADH2 → 3 ATP (via ETC)

2 GTP = 2 ATP (via substrate-level)

Total = 20 ATP JUST from TCA

For questions about ATP production, what is imporatnt to remember?

• One glucose → 2 pyruvate → 2 acetyl-coA → 2 TCA cycles

• Direct ATP vs. ATP from NADH/FADH2

  • Direct: 2 ATP from substrate-level via TCA

  • Indirect: ATP from NADH/FADH2

• DO NOT include ATP from glycolysis or PDH if asking about just “TCA”

What is the mnemonic to remember TCA products?

Can → Citrate (via citrate synthase)

I → Isocitrate (via aconitase)

Keep → alpha-ketoglutarate (Isocitrate dehydrogenase)

Selling → Succinyl CoA (alpha-ketoglutarate dehydrogenation complex)

Sex → Succinate (Succinyl CoA Synthetase)

For → Fumarate (Succinate Dehydrogenase)

Money → Malate (Fumarase)

Officer → Oxaloacetate (Malate Dehydrogenase)

Does the TCA have a “commitment” step>

no

What are the 3 control points of regulation in TCA?

Step 1 - Citrate Synthase

Acetyl CoA + Oxaloacetate —Citrate Synthase→ Citrate

Step 3 - Isocitrate Dehydrogenase

Isocitrate + NAD+ —Isocitrate dehydrogenase→ a-ketoglutarate + CO2.+ NADH

Step 4 - alpha-ketoglutarate dehydrogenase complex

a-ketoglutarate + NAD+ —a-ketoglutarate dehydrogenase complex→ Succinul CoA

What is the regulation at step 1

Step 1 - Citrate Synthase

Acetyl CoA + Oxaloacetate —Citrate Synthase→ Citrate

Inhibition:

• NADH, ATP

Both are indicative of high energy charge which inhibit the cycle

• bc no need to make more NADH which would make more energy because we already have high energy

What is the regulation at step 3 (activation and inhibition)

Step 3 - Isocitrate Dehydrogenase

Isocitrate + NAD+ —Isocitrate dehydrogenase→ a-ketoglutarate + CO2.+ NADH

Inhibition

• ATP NADH

• Inhibition: High energy charge slows cycle

Activation

• Calcium, ADP 

• Activation: Low energy charge stimulates cycle

Key fkux control point

What is the regulation at step 4

Step 4 - alpha-ketoglutarate dehydrogenase complex

a-ketoglutarate + NAD+ —a-ketoglutarate dehydrogenase complex→ Succinul CoA

Inhibition

• ATP/GTP & NADH (high energy charge), Succinyl CoA (product via negative feedback)

Inhibition of high eneryg charge and of succinyl CoA product prevents overproduction

Wh

What is a pattern that could be noticed with regulation of the TCA?

All regulation takes place in 1st half of cyccle

• Inhibiting firrst half inhibits 2nd half since the intermediates aren’t being produced

What is the catabolic reaction that TCA does (the overall rxn)

Acetyl-CoA → CO2 + ATP/GTP + NADH + FADH2

Which intermediates of TCA can be used for Anabolic processes (and what are they)

Citrate: Fatty acid + Sterol synthesis

a-ketoglutarate: Amino Acid synthesis (glutamate), purines

Succinyl-CoA: Heme synthesis

Oxaloacetate: Aspartate, asparagine synthesis

What happens if intermediates of the TCA are removed? What needs to be done after>

Cycle stops (because there is no final carbon product, each reaction relies on an intermediate)

• Need anaplerotic reaction to kickstart Cycle again

What is an anaplerotic reaction? What major enzyme does it for the TCA?

Anaplerotic rxn replenishes intermediates

• TCA: Pyruvate Carboxylase (from gluconeogenesis) replenishses Oxaloacetate

Pyruvate + CO2 + ATP —Pyruvate Carboxylase→ Oxaloacetate + ADP + Pi

What is pyruvate carboxylase activated by (allosteric activator)

INC Acetyl-CoA → INC Pyruvate Carboxylase activity → INC Oxaloacetate → INC TCA

What are other minor anaplerotic rxn?

Amino acid transamniations

PEP Carboxykinase (reverse direction reaction)

• Phosphoenolpyruvate → Oxaloacetate

• INC PeP —PEP carboxykinase (in reverse)→ INC Oxaloacetate in Cytoplasm → Transported into Mitochondria cia Malate-Aspartate Shuttle → INC TCA

Why is PEP Carboxykinase not as a major contributor to anaplerotic rxn as Pyruvate carboxylase?

Pyruvate carboxylase takes place in the mitochondria already, so it doesn’t need to be reformed to malate and re transported. Can immediately go into TCA

PEP carboxykinase takes place in cytoplasm so it  needs to transport

How does the TCA have diverse fuel input options with carbs, farry acids, and proteins?

All eventually produce acetyl-CoA

Carbs: Glucose → Pyruvate → ACetyl-CoA

• via PDH complex

Fatty Acids: B-oxidation → Acetyl-CoA

• major fuel sources, during fasting

Proteins: Amino Acids → Acetyl-CoA or other cycle intermediates 

• Starvation

What Are the products and intermediates of TCA used for?

NADH & FADH2: Energy via ETC to produce ATP

Citrate, a-ketoglutarate, succinyl-CoA, oxaloacetate for Biosynthesis

CO2 and H2O completly oxidized

How does THiamaine defciciency affect TCA?

Thiamine phosphoryate needed for PDH complex to function

• No PDH activity → DEC Acetyl-CoA → DEC TCA

How does Arsenic poisoniing affect TCA?

Arsenic binds to lipoamide → Inhibits a-ketoglutarate dehydrogenase complex

• Inhibition of a-ketoglutarate dehydrogenase complex causes backup buildup of intermediatess → Cant’ function

How does Cancer Metabolism affect the TCA? What is the specific effect called? How are TCA intermediates used?

Some tumors use TCA differently, called the Warburg effect

TCA intermediates (oncometabolites) succinate and fumarate accumulate

• beacuse no oxidative phosphorylation happens. They complete with de-methylating enzymes, preventing cells from expressing p53 gene to combat the cancer

WHat is the warburg effect?

Cancer cells prefer to use glycolysis (anarobic respiration) instead of oxidative phosphorylation (aerobic respiration)

• Even though not as efficient, cancer cells want the energy FAST as possible

Since cancer cells do mainly glycolysis and no oxidative, what happens with pyruvate?

Pyruvate accumulates, and is used as the carbon skeleton and building block for carbon structures in the cell