Bio 4A Citric Acid Cycle (Krebs Cycle) Worksheet illustrating the metabolic pathway with labeled molecules, intermediates, and enzymes.

The image you've provided is a Citric Acid Cycle (Krebs Cycle) Worksheet for a biology class. The diagram illustrates the steps of the citric acid cycle, with various intermediates and molecules labeled in boxes, and some missing labels that need to be filled in.

Let’s go step-by-step through the Citric Acid Cycle, identify each intermediate, molecule, and enzyme involved, and fill in the blanks accordingly.

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🔬 Overview of the Citric Acid Cycle:

- Begins with Acetyl-CoA combining with oxaloacetate to form citrate.
- A series of 8 steps involving oxidation, decarboxylation, and regeneration of oxaloacetate.
- Produces: 3 NADH, 1 FADH₂, 1 ATP (or GTP), and 2 CO₂ per Acetyl-CoA.

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We will now label each part based on standard biochemistry knowledge.

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🔄 Step-by-Step Analysis:

#### ✔ Starting Point:
- Acetyl-CoA enters the cycle.
- It combines with Oxaloacetate (a 4-carbon molecule) to form Citrate (6-carbon).

> 🔹 First blank next to Acetyl-CoA → Enzyme: Citrate Synthase

> 🔹 Intermediate after Acetyl-CoA + Oxaloacetate → Citrate

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#### 🔁 Step 1: Acetyl-CoA + Oxaloacetate → Citrate
- Enzyme: Citrate Synthase
- Molecule produced: Citrate
- So:
- Intermediate: Citrate

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#### 🔁 Step 2: Citrate → Isocitrate
- Isomerization via Aconitase
- Citrate → cis-Aconitate → Isocitrate
- No net change in carbon count; just rearrangement.

> 🔹 Intermediate: Isocitrate
> 🔹 Enzyme: Aconitase

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#### 🔁 Step 3: Isocitrate → α-Ketoglutarate
- Oxidative decarboxylation
- Isocitrate Dehydrogenase catalyzes this step
- Produces: NADH + H⁺, CO₂

> 🔹 Intermediate: α-Ketoglutarate
> 🔹 Molecule: NADH (yellow box)
> 🔹 Molecule: CO₂ (gray oval)

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#### 🔁 Step 4: α-Ketoglutarate → Succinyl-CoA
- Another oxidative decarboxylation
- α-Ketoglutarate Dehydrogenase Complex (similar to pyruvate dehydrogenase)
- Produces: NADH + H⁺, CO₂, Succinyl-CoA

> 🔹 Intermediate: Succinyl-CoA
> 🔹 Molecule: NADH (yellow box)
> 🔹 Molecule: CO₂ (gray oval)

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#### 🔁 Step 5: Succinyl-CoA → Succinate
- Succinyl-CoA Synthetase catalyzes substrate-level phosphorylation
- Produces ATP (or GTP) from GDP/ADP
- This is the only step in the cycle that directly produces ATP

> 🔹 Intermediate: Succinate
> 🔹 Molecule: ATP (or GTP) – yellow box
> 🔹 Enzyme: Succinyl-CoA Synthetase

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#### 🔁 Step 6: Succinate → Fumarate
- Oxidation by Succinate Dehydrogenase
- Produces FADH₂
- FAD is reduced to FADH₂

> 🔹 Intermediate: Fumarate
> 🔹 Molecule: FADH₂ (yellow box)
> 🔹 Enzyme: Succinate Dehydrogenase

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#### 🔁 Step 7: Fumarate → Malate
- Hydration by Fumarase
- Water added across double bond

> 🔹 Intermediate: Malate
> 🔹 Enzyme: Fumarase

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#### 🔁 Step 8: Malate → Oxaloacetate
- Oxidized by Malate Dehydrogenase
- Produces NADH + H⁺
- Regenerates Oxaloacetate to continue the cycle

> 🔹 Intermediate: Oxaloacetate
> 🔹 Molecule: NADH (yellow box)

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✔ Final Summary of Labels:

| Box Position | Label |
|-------------|-------|
| Top right: Enzyme for Acetyl-CoA + Oxaloacetate → Citrate | Citrate Synthase |
| First intermediate after Acetyl-CoA | Citrate |
| After Citrate | Isocitrate |
| After Isocitrate | α-Ketoglutarate |
| After α-Ketoglutarate | Succinyl-CoA |
| After Succinyl-CoA | Succinate |
| After Succinate | Fumarate |
| After Fumarate | Malate |
| Final intermediate | Oxaloacetate |
| Yellow boxes (Molecules): | NADH, NADH, NADH, FADH₂, ATP/GTP, CO₂, CO₂ |
| Gray ovals (CO₂) | CO₂, CO₂ |
| Enzyme between Succinyl-CoA and Succinate | Succinyl-CoA Synthetase |
| Enzyme between Succinate and Fumarate | Succinate Dehydrogenase |
| Enzyme between Fumarate and Malate | Fumarase |
| Enzyme between Malate and Oxaloacetate | Malate Dehydrogenase |

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💡 Key Notes:

- CO₂ is released in two steps: isocitrate → α-ketoglutarate and α-ketoglutarate → succinyl-CoA.
- NADH is produced 3 times: isocitrate, α-ketoglutarate, malate → oxaloacetate.
- FADH₂ once: succinate → fumarate.
- ATP/GTP once: succinyl-CoA → succinate.
- Oxaloacetate is regenerated at the end to restart the cycle.

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✔ Final Answer (Fill-in-the-blanks):

Here’s how to complete the worksheet:

```
[Intermediate:] Citrate
[Enzyme]: Citrate Synthase

[Intermediate:] Isocitrate
[Intermediate:] α-Ketoglutarate
[Molecule] NADH
[Molecule] CO₂

[Intermediate:] Succinyl-CoA
[Molecule] NADH
[Molecule] CO₂

[Intermediate:] Succinate
[Enzyme] Succinyl-CoA Synthetase
[Molecule] ATP (or GTP)

[Intermediate:] Fumarate
[Enzyme] Succinate Dehydrogenase
[Molecule] FADH₂

[Intermediate:] Malate
[Enzyme] Fumarase

[Intermediate:] Oxaloacetate
[Molecule] NADH
```

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📌 Bonus: Energy Yield per Acetyl-CoA

- 3 NADH → 3 × 2.5 = 7.5 ATP
- 1 FADH₂ → 1 × 1.5 = 1.5 ATP
- 1 ATP → 1 ATP
- Total ≈ 10 ATP per Acetyl-CoA

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