Comprehensive TCA Cycle and Pyruvate Dehydrogenase Complex Review for Biochemistry Students

Where does the transition from Pyruvate to Acetyl-CoA occur?

In the mitochondrial matrix.

What multi-enzyme complex catalyzes the conversion of Pyruvate to Acetyl-CoA?

The Pyruvate Dehydrogenase (PDH) Complex.

What are the three enzymes that make up the PDH complex?

$E_{1}$ (pyruvate dehydrogenase), $E_{2}$ (dihydrolipoyl transacetylase), and $E_{3}$ (dihydrolipoyl dehydrogenase).

Name the five essential cofactors required by the PDH complex.

TPP, Lipoic acid, CoA, FAD, and $NAD^{+}$.

What is the specific role of TPP in the PDH reaction?

It decarboxylates pyruvate, releasing $CO_{2}$ and forming a hydroxyethyl-TPP intermediate.

What is the function of the 'Lipoamide swinging arm' in $E_{2}$?

It picks up the acetyl group from $E_{1}$ and delivers it to Coenzyme A.

Which PDH subunit is responsible for regenerating the oxidized form of lipoamide?

$E_{3}$ (using FAD and $NAD^{+}$).

What are the three chemical products of the PDH complex reaction?

Acetyl-CoA, $CO_{2}$, and NADH.

How is the PDH complex regulated by its products?

It is inhibited by high levels of Acetyl-CoA and NADH.

Which enzyme phosphorylates (and thus inactivates) the PDH complex?

PDH Kinase.

What activates the PDH Phosphatase (reactivating the complex)?

$Ca^{2+}$ and insulin.

Why is the TCA cycle called 'amphibolic'?

It functions in both catabolism (breaking down fuel) and anabolism (providing precursors for biosynthesis).

What is the net yield of one turn of the TCA cycle?

3 NADH, 1 $FADH_{2}$, 1 GTP (or ATP), and 2 $CO_{2}$.

How many ATP equivalents are produced from one molecule of Acetyl-CoA after the ETC?

Approximately 10 ATP (3 NADH $ imes$ 2.5 + 1 $FADH_{2}$ $ imes$ 1.5 + 1 GTP).

Where are all TCA cycle enzymes located, except for one?

The mitochondrial matrix.

Which TCA enzyme is bound to the inner mitochondrial membrane?

Succinate Dehydrogenase (Complex II).

Which enzyme catalyzes the condensation of Acetyl-CoA and Oxaloacetate?

Citrate Synthase.

What is the first 6-carbon intermediate formed?

Citrate.

Why is Citrate Synthase considered a 'pacemaker' enzyme?

It is the first step and is highly exergonic, making it a key regulatory point.

What enzyme converts Citrate to Isocitrate?

Aconitase.

What is the intermediate formed during the Aconitase reaction?

cis-Aconitate.

Why must Citrate be isomerized to Isocitrate?

Tertiary alcohols (like citrate) cannot be oxidized; the hydroxyl must move to a secondary position.

What enzyme catalyzes the first oxidative decarboxylation?

Isocitrate Dehydrogenase.

What are the products of the Isocitrate Dehydrogenase reaction?

$eta$-Ketoglutarate, $CO_{2}$, and NADH.

Which molecule acts as an allosteric activator for Isocitrate Dehydrogenase?

ADP (signals low energy).

What enzyme catalyzes the second oxidative decarboxylation?

$eta$-Ketoglutarate Dehydrogenase ($eta$-KGDH).

What high-energy thioester is produced by $eta$-KGDH?

Succinyl-CoA.

Which complex is $eta$-KGDH structurally similar to?

The PDH Complex (uses the same 5 cofactors).

Which enzyme performs substrate-level phosphorylation in the TCA?

Succinyl-CoA Synthetase.

What is the high-energy product of this step?

GTP (which is readily converted to ATP).

Which enzyme oxidizes Succinate to Fumarate?

Succinate Dehydrogenase.

Why is FAD used instead of $NAD^{+}$ in this step?

The free energy change of succinate oxidation is insufficient to reduce $NAD^{+}$ but enough for FAD.

What enzyme hydrates Fumarate to L-Malate?

Fumarase.

What enzyme regenerates Oxaloacetate from Malate?

Malate Dehydrogenase.

Is the Malate Dehydrogenase reaction favorable under standard conditions?

No, it is highly endergonic ($+29.7$ kJ/mol), but proceeds because oxaloacetate is rapidly consumed by Step 1.

What are the three primary rate-determining steps of the TCA cycle?

Steps 1 (Citrate Synthase), 3 (Isocitrate Dehydrogenase), and 4 ($eta$-KGDH).

How does high [NADH] affect the cycle?

It inhibits the three rate-determining enzymes, slowing the cycle.

How does Succinyl-CoA regulate the cycle?

It acts as a competitive inhibitor for Citrate Synthase and $eta$-KGDH (feedback inhibition).

What effect does $Ca^{2+}$ have on the TCA cycle?

It activates Isocitrate Dehydrogenase and $eta$-KGDH (linking muscle contraction to energy production).

Why is the TCA cycle inhibited by high [ATP]?

High ATP signals a 'high energy charge,' indicating the cell does not need more fuel oxidation.

Define 'Anaplerotic Reaction.'

A reaction that replenishes intermediates of a metabolic pathway (e.g., replenishing oxaloacetate).

Which enzyme converts Pyruvate directly to Oxaloacetate?

Pyruvate Carboxylase.

What allosteric activator is required by Pyruvate Carboxylase?

Acetyl-CoA.

Which TCA intermediate is used for Heme synthesis?

Succinyl-CoA.

Which TCA intermediate is used for Fatty Acid synthesis?

Citrate (it is exported to the cytosol).

Which TCA intermediate is the precursor for Gluconeogenesis?

Oxaloacetate.

$eta$-Ketoglutarate is a precursor for which amino acid family?

The Glutamate family.

Oxaloacetate is a precursor for which amino acid family?

The Aspartate family.

Which poison inhibits Aconitase?

Fluoroacetate (converted to fluorocitrate).

What is Fluoroacetate converted to?

Fluorocitrate.

Which heavy metal binds to lipoic acid, inhibiting PDH and α-KGDH?

Arsenite (AsO₃³⁻).

What is the Glyoxylate Cycle?

A variation of the TCA cycle in plants and bacteria that allows for the net synthesis of glucose from Acetyl-CoA.

What two enzymes are unique to the Glyoxylate Cycle?

Isocitrate Lyase and Malate Synthase.

Is Citrate a chiral molecule?

No, it is achiral (symmetrical).

If Citrate is achiral, how does Aconitase only produce L-Isocitrate?

Aconitase treats Citrate as 'prochiral' because it binds the substrate at three specific points.

What is the 'Pro-R' and 'Pro-S' arm of Citrate?

Labels for the two identical-looking CH₂COO⁻ groups that the enzyme can distinguish.

How many carbons are lost as CO₂ in one full turn of the cycle?

Two carbons.

Are the carbons lost as CO₂ the same ones that entered as Acetyl-CoA in that specific turn?

No, they come from the Oxaloacetate backbone.

What type of bond links the acetyl group to Coenzyme A?

A high-energy thioester bond.

What is the reactive part of Coenzyme A?

The thiol group (-SH) at the end of β-mercaptoethylamine.

Which vitamin is Coenzyme A derived from?

Pantothenic acid (B₅).

Per molecule of glucose, how many NADH are produced by the TCA cycle alone?

6 NADH.

Per molecule of glucose, how many CO₂ are produced from the bridge step plus TCA?

6 CO₂.

Why does the TCA cycle stop in the absence of Oxygen?

Without O₂, the ETC cannot regenerate NAD⁺ and FAD from NADH and FADH₂.

What is the difference between a 'Synthase' and a 'Synthetase'?

Synthetases require a nucleoside triphosphate (ATP/GTP); Synthases do not.

What is the ΔG°' for the Citrate Synthase reaction?

-31.5 kJ/mol (highly favorable).

What is the ΔG°' for the Isocitrate Dehydrogenase reaction?

-21 kJ/mol.

What is the ΔG°' for the α-Ketoglutarate Dehydrogenase reaction?

-33 kJ/mol.

Which reaction produces the first CO₂ of the cycle?

Isocitrate Dehydrogenase.

What did Albert Szent-Gyorgyi discover regarding the TCA cycle?

That Succinate → Fumarate → Malate → Oxaloacetate was a sequence that stimulated O₂ consumption.

What was Hans Krebs' major contribution in 1937?

He realized the pathway was a cycle, not a linear chain, by showing Citrate is formed from Oxaloacetate and Pyruvate.

Which inhibitor did Krebs use to prove the cycle?

Malonate (which inhibits Succinate Dehydrogenase).

What happens when Malonate is added to a cell?

Succinate accumulates and the cycle stops.

What is the role of NADH/NAD ratio in the cycle?

High ratio = Cycle Off; Low ratio = Cycle On.

Does Glucagon directly regulate TCA cycle enzymes?

Generally no; it regulates the supply of substrates (like glucose/fatty acids).

Which enzyme is part of the iron-sulfur (Fe-S) protein family?

Aconitase.

What happens to the Acetyl-CoA if the cell has plenty of ATP?

It is diverted to fatty acid synthesis.

How many steps of the TCA cycle involve oxidation?

Four steps (Steps 3, 4, 6, and 8).

Which enzyme produces the FADH₂?

Succinate Dehydrogenase.

What is the relationship between α-Ketoglutarate and the Urea cycle?

It accepts amino groups to become Glutamate, facilitating nitrogen removal.

What is the byproduct of the Succinyl-CoA Synthetase reaction besides GTP?

Free Coenzyme A (CoA-SH).

Which reaction involves the addition of H₂O?

Fumarase.

What is the 'catalytic' intermediate of the cycle?

Oxaloacetate (it is used and then regenerated).

If one oxaloacetate is added to a system, how many Acetyl-CoA can it help oxidize?

Theoretically, an infinite amount (as long as it is regenerated).

What is the role of the E3 subunit in the PDH complex?

It uses FAD to re-oxidize the lipoamide swinging arm.

What is the final electron acceptor for the electrons taken by E₃?

NAD⁺.

Which PDH subunit uses TPP?

E₁.

What does the term 'Dehydrogenase' typically imply?

An oxidation-reduction reaction involving NAD⁺ or FAD.

Is the TCA cycle efficient?

Yes, it captures about 90% of the energy available from oxidation in its intermediates.

Which intermediate is a 5-carbon molecule?

α-Ketoglutarate.

How many 4-carbon intermediates are in the cycle?

Four (Succinate, Fumarate, Malate, Oxaloacetate) - or five if counting Succinyl-CoA.

What is the standard concentration of Oxaloacetate in the mitochondria?

Extremely low (micromolar range).

Why is low [Oxaloacetate] beneficial?

It makes the Malate Dehydrogenase reaction possible by pulling the product toward Citrate Synthase.

Which step is inhibited by high [Citrate]?

PFK-1 in Glycolysis (coordinating the two pathways).

What is the symmetry of Fumarate?

It is a symmetrical 4-carbon dicarboxylic acid with a double bond.

Can mammals convert Acetyl-CoA into Glucose?

No, because the two carbons entering as Acetyl-CoA are lost as CO₂ before they can reach Oxaloacetate for gluconeogenesis.

Which enzyme is inhibited by high levels of Acetyl-CoA?

Pyruvate Dehydrogenase (E₁).

Which intermediate is the precursor to Porphyrins (like in hemoglobin)?

Succinyl-CoA.

Does the TCA cycle produce any ATP directly?

Technically, it produces GTP, which is an ATP equivalent.

What is the 'hub' of metabolism?

The Citric Acid Cycle.