Biochem Lect 31: TCA Cycle

Aerobic Metabolism

-

grow only without O₂/ROS, use fermentation for energy only

obligate anaerobes

can handle presence of O₂/ROS, use fermentation for energy only

aerotolerant anaerobes

(tolerant, don’t use O₂)

can handle presence of O₂/ROS, can use O₂/ROS for energy if available

facultative anaerobes

(use O₂)

dependent on O₂

obligate aerobes

aerotolerant/facultative anaerobes and obligate aerobes contain ___________ to handle O₂

detoxifying enzymes (detoxify ROS)

Which 3 processes are considered part of aerobic metabolism?

TCA cycle, ETC, oxidative phosphorylation

Aerobic metabolism (including TCA cycle) usually occurs in cytoplasm or mitochondria?

mitochondria

electrons are transferred to someone with higher _______ potential

reduction (reduce = gain e-)

2 main categories of redox coenzymes

1) NAD/NADPH

2) FAD/FMN

NAD = ?

NADPH = ?

NAD = nicotinamide adenine dinucleotide

NADPH = nicotinamide adenine dinucleotide phosphate

NAD/NADPH structure - both contain:

• _______

• nicotinamide N-ribosyl derivative

• linked thru _______

NAD/NADPH structure - both contain:

• adenosine

• nicotinamide N-ribosyl derivative

• linked thru pyrophosphate

FAD = ?

FMN = ?

FAD = flavine adenine dinucleotide

FMN = flavin mononucleotide

FAD/FMN structure

________ (_________) are tightly bound prosthetic groups in _________ (FAD/FMN).

FAD/FMN structure

Riboflavin (Vitamin B₂) are tightly bound prosthetic groups in flavoenzymes (FAD/FMN).

NADPH carries ___ electrons and transfers ___ at a time.

FAD/FMN carries ___ electrons and tranfers ___ at a time.

NADPH carries 2 electrons and transfers 2 at a time.

FAD/FMN carries 2 electrons and transfers 1 or 2 at a time.

TCA/Citric Acid/Krebs Cycle

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Overall reactants and products of TCA cycle

Acetyl CoA + Oxaloacetate → energy (NADH₂, FADH₂)

Acetyl Coa can be made in many ways, for example…

• from pyruvate !!

• fatty acid catabolism

• some reactions in amino acid metabolism

TCA Cycle 2 phases overall (_ reactions)

1) acetyl-CoA + Oxaloacetate → ______

2) ??

TCA Cycle 2 phases overall (8 reactions)

1) acetyl-CoA + Oxaloacetate → 2 CO₂ released

2) Oxaloacetate is regenerated

Phase I has ___ reactions, Phase II has ___ reactions

5, 3

What are the regulated steps of TCA cycle?

1, 3, 4

Phase I

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Summary of Phase I (Reactants → Products)

1) Acetyl-CoA + Oxaloacetate → Citrate

2) Citrate → Isocitrate

3) Isocitrate → ɑ-Ketoglutarate

4) ɑ-Ketoglutarate → Succinyl CoA

5) Succinyl-CoA → Succinate

Summary of Phase I (Enzymes)

1) Citrate Synthase

2) Aconitase

3) Isocitrate Dehydrogenase

4) ɑ-Ketoglutanate Dehydrogenase Complex

5) Succinyl-CoA Synthetase

Summary of Phase I (Extras)

1) -ΔG

2)

3) +CO₂, +NADPH/H+, -ΔG

4) +CO₂, +NADPH/H+, +CoA-SH

5) +GTP, -CoA-SH

Step 1: ________ → ________ (E = ________)

• -

• -

Step 1: Acetyl-CoA + Oxaloacetate → Citrate (E = Citrate Synthase)

• aldol condensation + hydrolysis

• -ΔG (regulated)

Step 2: ________ → ________ (E = ________)

• -

• -

Step 2: Citrate → Isocitrate (E = Aconitase)

• dehydration/rehydration

• intermediate step involving cis-Aconitate

Aconitase removes the pro R/S H arm of citrate via a ______

R, Fe-S cluster

Aconitase contains a ___Fe-___S cluster bound to a protein by 3 ___.

Inactive form = ___Fe-___S (binding ____ activates aconitase)

Aconitase contains a 4Fe-4S cluster bound to a protein by 3 Cys.

Inactive form = 3Fe-4S (binding Fe²⁺ activates aconitase)

Step 3: ________ → ________ (E = ________)

• -

• -

Step 3: Isocitrate → ɑ-Ketoglutarate (E = Isocitrate Dehydrogenase)

• oxidative decarboxylation (+CO₂, +NADPH/H+)

• -ΔG (regulated)

Step 3 mechanism (oxidative decarboxylation):

1) isocitrate —oxidized—> ____(1)____

2) ____(1)____ —decarboxylated—> ____(2)____

3) ____(2)____ —> ɑkg

Step 3 mechanism (oxidative decarboxylation):

1) isocitrate —oxidized—> oxalosuccinate

2) oxalosuccinate —decarboxylated—> enol intermediate

3) enol intermediate —> ɑkg

Step 4: ________ → ________ (E = ________)

• -

• -

Step 4: ɑ-Ketoglutarate → Succinyl CoA (E = ɑ-Ketoglutarate Dehydrogenase Complex)

• 2nd oxidative decarboxylation (+CO₂, +NADPH/H+)

• add CoA-SH

ɑkg dehydrogenase complex involves what 3 enzymes?

1) ɑkg dehydrogenase

2) dihydrolipoyl transuccinylase

3) dihydrolipoyl dehydrogenase

Step 5: ________ → ________ (E = ________)

• -

• -

• -

Step 5: Succinyl-CoA → Succinate (E = Succinyl-CoA Synthetase)

• +GTP (synthetase)

• remove CoA-SH

• coupled with SLP

Phase II

-

Summary of Phase II (Reaction Name)

6) Oxidation (single → double bond)

7) Trans-Hydration

8) Oxidation (OH → ketone)

Summary of Phase II (Reactants → Products)

6) Succinate → Fumarate

7) Fumarate → Malate

8) Malate → Oxaloacetate

Summary of Phase II (Enzymes)

6) Succinate Dehydrogenase

7) Fumarase

8) Malate Dehydrogenase

Summary of Phase II (Extras)

6) +FADH₂

7) -H₂O

8) +NADH

Step 6: ________ → ________ (E = ________)

• -

• -

Step 6: Succinate → Fumarate (E = Succinate Dehydrogenase)

• trans-oxidation = oxidation of single to double bond

• +FADH₂

FAD is covalently bound to ___________________.

The ___ carbon of FAD is linked to ____ of this enzyme.

FAD is covalently bound to succinate dehydrogenase.

The C8a carbon of FAD is linked to His of this enzyme.

Succinate dehydrogenase has 3 types of Fe-S clusters…

1) 2Fe-2S

2) 3Fe-4S

3) 4Fe-4S

Fe-S clusters are “redox centers” that participate in ___ e- redox rxns (involving ___ and ___ oxidation states)

Fe-S clusters participate in 1 e- redox rxns (involving Fe²⁺ and Fe³⁺ oxidation states)

_________ = multiple redox centers close to each other (makes redox rxns easier)

electron wire

Step 7: ________ → ________ (E = ________)

• -

Step 7: Fumarate → Malate (E = Fumarase)

• trans-hydration (-H₂O) = hydration across double bond

Step 8: ________ → ________ (E = ________)

• -

Step 8: Malate → Oxaloacetate (E = Malate Dehydrogenase)

• OH → ketone oxidation (+NADH)

TCA Cycle Yield

-

oxidation of glucose to CO₂ is a ___ e- oxidation

oxidation of glucose to CO₂ is a 24 e- oxidation

Products of 1x TCA Cycle

• ___ CO₂

• ___ NADH

• ___ FADH₂

• ___ ATP

Products of 1x TCA Cycle

• 2 CO₂

• 3 NADH

• 1 FADH₂

• 1 ATP

Products of 1x TCA Cycle + Glycolysis

• ___ CO₂

• ___ NADH

• ___ FADH₂

• ___ ATP

Products of 1x TCA Cycle + Glycolysis

• 6 CO₂

• 10 NADH

• 2 FADH₂

• 4 ATP

10 NADH + 2 FADH₂ → ___ ATP

10 NADH + 2 FADH₂ → 34 ATP (varies)

TCA Cycle is at the center ________ and ________ pathways

catabolic, anabolic

anaplerotic reactions

“filling up” reactions

(replenish TCA cycle intermediates)

carbon source of anaplerotic reactions

CO₂

The most important anaplerotic enzyme is ________ which catalyzes ________ → ________, providing a link between glycolysis and the TCA cycle.

The most important anaplerotic enzyme is pyruvate carboxylase which catalyzes pyruvate → oxaloacetate, providing a link between glycolysis and the TCA cycle.

pyruvate carboxylase is activated by ________.

acetyl-CoA

(if acetyl CoA > oxaloacetate, more oxaloacetate will be made)

TCA Cycle regulation (steps ___, ___, ___ + _________ reaction)

• ATP/AMP/ADP inhibits/activates

• NADH/NAD+ inhibits/activates

• succinyl CoA inhibits/activates

TCA Cycle regulation (steps 1, 3, 4 + pyruvate dehydrogenase reaction)

• ATP inhibits, AMP activates

• NADH inhibits, NAD+ activates

• succinyl CoA inhibits

NAD+ = high/low energy signal, NADH = high/low energy signal

ATP = high/low energy signal, AMP/ADP = high/low energy signal

succinyl CoA = signal that __________

NAD+ = low energy signal, NADH = high energy signal

ATP = high energy signal, AMP/ADP = low energy signal

succinyl CoA = signal that cycle is saturated

-

*NADH = High

Pyruvate Dehydrogenase Regulation

-

PDH complex catalyzes what reaction?

pyruvate → acetyl CoA

PDH complex contains what 3 enzymes?

1) pyruvate dehydrogenase

2) dihydrolipoyl transacetylase

3) dihydrolipoyl dehydrogenase

_______________ inactivates PDHC in 2 ways: ________, __________

PDH kinase inactivates PDHC in 2 ways: allosterically, serine phosphorylation

PDH kinase is activated by ________ and ________

NADH and acetyl CoA

(opposite of PDH Complex)

_______________ activates PDHC

PDH phosphatase activates PDHC