SECTION 05: ENZYME INHIBITION

Q: Why does the cell need to regulate enzyme activity?

A: To meet metabolic needs, prevent overproduction of products, and avoid shortages of essential molecules.

Q: How is enzyme activity regulated in the cell?

A: Through enzyme regulation methods and environmental/physical factors.

Q: What type of inhibitors are the main focus of this section?

A: Reversible inhibitors mainly and a little bit of irreversible inhibitors (briefly)

What are reversible enzyme inhibitors?

Reversible inhibitors temporarily reduce enzyme activity without permanently damaging the enzyme.

What are the 3 main types of reversible inhibitors?

1. Competitive

2. Uncompetitive

3. Non-competitive

Competitive Inhibitor (CI)

• Binds at the active site

• Competes with the substrate

• Km ↑ (substrate needs to outcompete inhibitor)

• Vmax stays the same

• Trick: "Competitive = Km increases"

• Lineweaver-Burk: Lines intersect on y-axis

Non-competitive Inhibitor (NCI)

• Binds to free enzyme and ES complex (not at active site)

• Vmax ↓

• Km unchanged

• Trick: “Non-comp = No change in Km”

• Lineweaver-Burk: Lines cross at x-axis

What is the purpose of enzyme inhibitors?

They regulate enzyme activity to maintain balance—preventing too much or too little of a product from forming.

Types of Reversible Inhibitors (4)

• Competitive

• Uncompetitive

• Noncompetitive

• Mixed

What is Ki and Ki′?

• Ki = affinity of inhibitor for the free enzyme

• Ki′ = affinity of inhibitor for the enzyme-substrate (ES) complex
  → Smaller values = stronger inhibition

What is IC₅₀?

The drug concentration that reduces enzyme activity by 50% in vitro.

When are substrate-velocity curves used?

To compare how enzyme speed changes across different inhibitor concentrations—one curve must be without an inhibitor (control).

How does inhibition change the Michaelis-Menten equation?

What do the new terms mean?

• [I] = Inhibitor concentration

• Ki = Inhibitor’s affinity for free enzyme

• Ki′ = Inhibitor’s affinity for enzyme-substrate complex

What’s the meaning of the equation change?

• If [I] increases, the denominator gets bigger

• This slows the reaction rate (V₀)

• Whether Km or Vmax change depends on inhibitor type (covered in upcoming slides)

What does the left image show?

A competitive inhibitor (I) and a substrate (S) are both trying to bind to the enzyme’s active site (E).

• If S binds → ES complex → product

• If I binds → blocks substrate → no reaction

What does the right diagram represent?

It’s a visual of the reaction:
E + S ⇌ ES → E + P
↕

• I ⇌ EI

Where Ki is the binding affinity of the inhibitor.

Can the inhibition be reversed?

✅ Yes!
Adding more substrate (S) outcompetes the inhibitor → reaction resumes.

How do you increase ES complex formation?

By increasing either:

• The substrate

• The enzyme concentration

This shifts the balance toward product formation.

What do competitive inhibitors do?

• Competitive inhibitors compete with the substrate for the enzyme’s active site.

• They block the substrate from binding by fitting into the same site.

competitive inhibitors and enzyme kinetics - 📈 Flashcard 2: How do they affect Km and Vmax?

• Km increases (you need more substrate to reach half Vmax).

• Vmax stays the same (at high substrate concentration, the substrate can outcompete the inhibitor).

competitive inhibitors and enzyme kinetics - What happens on the Michaelis-Menten plot?

• Without inhibitor: Curve reaches Vmax quickly.

• With inhibitor: Curve is shifted right (takes more [S] to reach the same velocity).

• The top (Vmax) is the same across curves, but they rise more slowly with more inhibitor.

competitive inhibitors and enzyme kinetics - What does the Lineweaver-Burk plot show?

• Lines intersect at the Y-axis (Vmax unchanged).

• Slope increases with inhibitor = Line gets steeper.

• The X-intercept (−1/Km) shifts closer to zero = Higher Km.

• Mnemonic: "Competitive = Crosses at Y."

competitive inhibitors and enzyme kinetics - Key takeaway

• Competitive inhibitors make it harder for the substrate to bind (↑Km),
  but don't change the max speed of the reaction (Vmax stays the same).

What happens to Vmax in competitive inhibition?

• Vmax stays the same.

• Why? Because if you add enough substrate, it can outcompete the inhibitor for the active site.

competitive inhibition - What happens to Km?

• Km increases.

• Why? You need more substrate to reach 50% of Vmax when an inhibitor is present.

competitive inhibition - What causes the increase in Km?

• The Ki (inhibitor constant) and [I] (inhibitor concentration) together affect the apparent Km.

• Formula:

Reaction overview (diagram)

• The substrate (S) and inhibitor (I) both compete to bind to the enzyme (E).

• The enzyme can either form ES (productive) or EI (blocked).

• Only ES → E + P leads to product.

Key takeaway

• Competitive inhibitors don’t affect Vmax, but increase Km.

• The higher the [I], the more substrate you need to compete.

Activity

📊 Michaelis-Menten Plot

📈 Lineweaver-Burk Plot (Double Reciprocal Plot)

🧠 Use Case:

• Michaelis-Menten plot is better for visualizing enzyme behavior.

• Lineweaver-Burk plot is better for calculating exact Km and Vmax.

• Both help identify effects of enzyme inhibitors (e.g. competitive inhibition increases Km, Lineweaver-Burk shows this as steeper slope).

🧪 Uncompetitive Inhibitors – Binding

• Only bind after the substrate is bound (binds to enzyme-substrate complex (ES))

• Binds at an allosteric site, not the active site (but often near it)

• Binding prevents catalysis → enzyme becomes inactive

• Reduces the amount of available active enzyme

Uncompetitive Inhibitors – Binding

When do they bind?
➡ Only bind to the enzyme-substrate complex (ES) (not free enzyme)

Where do they bind?
➡ Bind at a site other than the active site (often near it)

What happens when they bind?
➡ Block enzyme activity – the enzyme can no longer convert substrate to product
➡ Reduces the number of active enzymes available

🔑 Key Idea:

Uncompetitive inhibitors don’t prevent binding of the substrate — they prevent the enzyme from doing its job after binding has already happened.

What are uncompetitive inhibitors?

Q: When do uncompetitive inhibitors bind?
A: Only after the substrate is bound to the enzyme (they bind the enzyme-substrate complex, not the free enzyme).

🟩 Flashcard 2: What happens to Km and Vmax in uncompetitive inhibition?

Q: How does an uncompetitive inhibitor affect Km and Vmax?
A: Both Km decreases and Vmax decreases.

🟩 Flashcard 3: Why does Km decrease?
Q: Why does apparent Km decrease in uncompetitive inhibition?

A: Because the enzyme is "trapped" in the ES complex, which increases its apparent affinity for the substrate.

🟩 Flashcard 4: Why does Vmax decrease?
Q: Why does Vmax decrease in uncompetitive inhibition?

A: The inhibitor prevents the ES complex from forming product, reducing the number of active enzymes.

🟩 Flashcard 5: Michaelis-Menten Plot (left graph)
Q: How does the Michaelis-Menten curve change with uncompetitive inhibition?

A: The curve shifts down (lower Vmax) and left (lower Km). More inhibition = more flattening and left shift.

🟩 Flashcard 6: Lineweaver-Burk Plot (right graph)
Q: What happens to the Lineweaver-Burk plot in uncompetitive inhibition?

A: The lines are parallel (same slope), shifted upward and left (because both 1/Vmax and -1/Km increase in magnitude).

🟩 Flashcard 7: Why are uncompetitive inhibitors more effective at high [S]?
Q: When are uncompetitive inhibitors most effective?

A: When there is lots of substrate, because they need the ES complex to form first.

🟦 Flashcard 1: Binding behavior
Q: Where do uncompetitive inhibitors bind?

A: They only bind to the enzyme-substrate complex (ES), not the free enzyme.

🟦 Flashcard 2: What happens to Km?
Q: What does uncompetitive inhibition do to Km?

A: It decreases Km (makes the enzyme seem to bind substrate better).

🟦 Flashcard 3: What happens to Vmax?
Q: What does uncompetitive inhibition do to Vmax?

A: It decreases Vmax by reducing the number of active enzymes available to make product.

🟦 Flashcard 4: Why is Km lowered?
Q: Why does Km decrease with uncompetitive inhibition?

A: Because the enzyme is trapped in the ES complex, making it appear to have higher substrate affinity.

🟦 Flashcard 5: When is uncompetitive inhibition most effective?
Q: When do uncompetitive inhibitors work best?

A: At high substrate concentrations, since more ES complexes are formed.

uncompetitive - 🟦 Flashcard 6: Equation overview
Q: What’s the uncompetitive Michaelis-Menten equation?

uncompetitive - 🟦 Flashcard 7: Reaction diagram meaning
Q: What does the reaction diagram show?

A:

• E + S forms ES

• Inhibitor (I) binds only to ES → forms ESI, which is inactive

• The enzyme cannot convert substrate to product from ESI

Binding Site
Q: Where do noncompetitive inhibitors bind?

A: To an allosteric site, not the active site.

🟩 Flashcard 2: Binding Target
Q: What can noncompetitive inhibitors bind to?

A: They bind equally well to the free enzyme [E] and the enzyme-substrate complex [ES].

🟩 Flashcard 3: Key Difference
Q: How are noncompetitive inhibitors different from competitive and uncompetitive inhibitors?

A:

• Competitive binds only E

• Uncompetitive binds only ES

• Noncompetitive binds both E and ES

Flashcard 1: Binding Targets
Q: What does a noncompetitive inhibitor bind to?

A: It binds equally well to the free enzyme (E) and the enzyme-substrate complex (ES).

🟪 Flashcard 2: Effect on Enzyme Function
Q: What happens when a noncompetitive inhibitor binds to the enzyme?

A: The enzyme’s ability to bind or release substrate is reduced, making it less catalytically active.

🟪 Flashcard 3: Main Outcome
Q: What is the result of noncompetitive inhibitor binding on enzyme concentration?

A: It lowers the effective enzyme concentration in the reaction—even if some enzyme is present, it's inactive when the inhibitor is bound.

Flashcard 1: What do noncompetitive inhibitors bind to?

🧬 Both the free enzyme (E) and the enzyme-substrate complex (ES)
✅ With equal affinity
🧷 Binding occurs at an allosteric site, not the active site

Flashcard 2: What is the effect of noncompetitive inhibitors on Km and Vmax?

📉 Vmax decreases
➖ Km stays the same
🧠 Because binding doesn't affect substrate binding, just catalytic activity

Flashcard 3: Michaelis-Menten Plot Explanation (Image 2)

🟢 As inhibitor concentration increases:
 • Max rate (Vmax) drops lower
 • Curve flattens earlier
📊 All lines still reach the same Km (same x-axis position), but at lower velocities

Flashcard 4: Lineweaver-Burk Plot Explanation (Image 1)

📈 Slope increases (steeper line)
🔹 Y-intercept (1/Vmax) goes up
➖ X-intercept (-1/Km) stays the same
💡 Confirms: Vmax ↓, Km unchanged

Flashcard 5: Key Takeaway

🛑 Noncompetitive inhibitors reduce enzyme efficiency, but don’t interfere with substrate binding
🧪 Used to shut down enzymes without affecting their substrate affinity

Flashcard 1: What is noncompetitive inhibition?

🔒 Inhibitor binds to both:

• Free enzyme (E)

• Enzyme-substrate complex (ES)
  📍 Binding occurs at a separate allosteric site

Flashcard 2: What happens to Km in noncompetitive inhibition?

➖ Km stays the same
📊 Why? Because the inhibitor binds both E and ES equally, keeping substrate binding affinity unchanged

Flashcard 3: What happens to Vmax in noncompetitive inhibition?

📉 Vmax decreases
🧪 Some enzyme is always blocked, so less is available to turn substrate into product

Flashcard 4: Michaelis-Menten equation with noncompetitive inhibitor

Flashcard 5: Visual interpretation (image)

🔁 E + I ↔ EI
🔁 ES + I ↔ ESI (inactive)
🛑 ESI cannot produce product → this reduces the overall reaction rate

Flashcard 1: What are Mixed Inhibitors?

🔀 Bind to both:

• Free enzyme (E)

• Enzyme-substrate complex (ES)
  🧩 Do not follow competitive, uncompetitive, or noncompetitive models exactly

Flashcard 2: Effect on Vmax

📉 Vmax always decreases
🧪 Because some enzyme is always inactivated, regardless of where the inhibitor binds

Flashcard 3: Effect on Km

🔼🔽 Km may increase or decrease
📊 Depends on the specific enzyme-inhibitor interaction
➡ Km behavior is variable in mixed inhibition

Flashcard 4: Summary Rule

Mixed inhibitors:

• 📉 Decrease Vmax

• 🔼 or 🔽 Change Km (depends on the system)

Flashcard 1: What do Mixed Inhibitors bind to?

🔗 Mixed inhibitors bind to:

• Free enzyme (E)

• Enzyme-substrate complex (ES)

Flashcard 2: What effect do Mixed Inhibitors have?

📉 Reduce the enzyme’s ability to:

• Bind substrate

• Release product
  ➡ This lowers the number of effective enzymes

Flashcard 3: Can product still be made?

✅ Yes, but at a slower rate
🧪 Product can be made from the EIS complex
📉 This slower rate is represented by k₀
🧠 In the math, this is called β (beta)

Flashcard 4: What’s unique about mixed inhibition vs noncompetitive?

⚖ Noncompetitive = equal affinity for E and ES
🔀 Mixed = different affinity for E and ES
📊 Result: More complex effect on kinetics

Q: What are the effects of mixed inhibitors on enzyme kinetics?

A: Mixed inhibitors always decrease Vmax, and Km may increase or decrease, depending on whether the inhibitor prefers free enzyme or ES complex.

Q: What does it mean when Km is decreased and Vmax is decreased (purple line)?

A: The inhibitor prefers the ES complex more.
🔹 This mimics uncompetitive inhibition.
🔹 Line intersects away from axes in Lineweaver-Burk.

Q: What does it mean when Km is increased and Vmax is decreased (red line)?

A: The inhibitor prefers the free enzyme more.
🔹 This mimics competitive inhibition.
🔹 Again, the Lineweaver-Burk lines intersect off-axis (not on x or y).

Q: On a Michaelis-Menten plot, how does mixed inhibition appear?

A:
🔸 Vmax shifts lower.
🔸 Depending on Km change, the curve is shifted left (↓Km) or right (↑Km).

Q: On a Lineweaver-Burk plot, how does mixed inhibition appear?

A:
🔸 Lines intersect off-axis (not at the y- or x-intercept).
🔸 Indicates both Km and Vmax are altered.

Q: What is the general equation used when the type of inhibition is unknown?

Q: What does K_i​ represent in mixed inhibition?

Ki​ is the affinity of the inhibitor for the free enzyme (E).

Q: What does Ki′​ represent in mixed inhibition?

A:
Ki′​ is the affinity of the inhibitor for the enzyme-substrate complex (ES).

Q: What does the ratio of Ki′ to Ki​ tell us?

A:
It tells us how much the inhibitor changes the enzyme’s affinity for the substrate. This ratio helps define the exact inhibition behavior.

Q: Why is the mixed inhibition model useful in experiments?

A:
It provides the best fit when the inhibition mechanism is uncertain or complex, covering multiple types in one formula.

Q: What is another name for irreversible inhibition?

Suicide inhibition — because the enzyme essentially destroys itself by reacting permanently with the inhibitor.

Q: How does irreversible inhibition work?

A:
The inhibitor forms a permanent covalent bond with the enzyme, usually at the active site.

Q: What kind of molecule is an irreversible inhibitor often similar to?

A:
A substrate analogue — it looks like the normal substrate and binds to the active site.

Q: What happens after the enzyme binds the irreversible inhibitor?

The enzyme modifies the inhibitor during the normal catalytic reaction, which activates a reactive group that irreversibly binds to the enzyme.

Q: What is the final result of irreversible inhibition?

A stable enzyme-inhibitor complex is formed, permanently inactivating the enzyme.

Q: What are two examples of drugs that act as irreversible inhibitors?

Aspirin and penicillin.

Q: What does penicillin target in bacteria?

It targets penicillin-binding protein (PBP), which is needed to cross-link peptide chains in the bacterial cell wall.

Q: What is the role of PBP in bacteria?

PBP binds short peptide chains (attached to NAM/NAG sugars) and links them to build a strong cell wall, then dissociates after the job is done.

Q: How does penicillin inhibit PBP?

Penicillin enters the PBP active site and reacts with a critical serine residue needed for enzyme activity.

Q: What happens to penicillin’s β-lactam ring during inhibition?

The β-lactam ring opens irreversibly and forms a covalent bond with PBP, permanently blocking the active site.

Q: Why is penicillin considered an irreversible inhibitor?

Because it forms a permanent covalent bond with the enzyme (PBP), stopping it from ever functioning again.

Q: What kind of drugs are statins and what do they target?

Statins are competitive inhibitors that block HMG-CoA reductase, the enzyme responsible for the rate-limiting step in cholesterol synthesis.