Catalytic Strategies 2

🌬 CO₂ Removal & Role of Carbonic Anhydrase

Why CO₂ Removal Matters:

• CO₂ must be removed from the body and replaced with O₂.

• CO₂ is not very soluble in blood → converted into carbonic acid (H₂CO₃) and bicarbonate (HCO₃⁻) for transport.

• In the lungs, it converts back to CO₂ for exhalation.

CO₂ Hydration Kinetics (Without Enzyme):

• CO₂ + H₂O ⇌ H₂CO₃ (slow: k₁ = 0.15 s⁻¹)

• H₂CO₃ ⇌ CO₂ + H₂O (faster: k₋₁ = 50 s⁻¹)

• Raising pH (to increase OH⁻) would speed it up, but that's not safe in blood.

• Solution: Use an enzyme → Carbonic Anhydrase.

🧬 Carbonic Anhydrase (CA)

Key Roles:

• 10% of CO₂ dissolves in plasma

• 20% binds to hemoglobin

• 70% converted to bicarbonate by CA

• In lungs: Bicarbonate → CO₂ (exhaled)

Enzyme Facts:

• Zn²⁺-containing enzyme

• Found in 7 gene families across life

• Found in the eye, bone formation, and brain

⚙ Mechanism of Carbonic Anhydrase

Active Site Structure:

• Zn²⁺ coordinated by:

  • 3 Histidine residues

  • 1 water (or hydroxide, depending on pH)

Catalysis Steps:

1. Zn²⁺ reduces water's pKa from 15.7 to 7 → generates OH⁻

2. CO₂ binds next to Zn²⁺

3. OH⁻ attacks CO₂ → forms HCO₃⁻

4. Water replaces HCO₃⁻ → resets active site

Buffer & Proton Shuttle:

• His64 shuttles proton to buffer

• Prevents back reaction (reprotonation)

• Buffer increases reaction rate by aiding proton removal

• Rate limited by proton diffusion, not CO₂ binding

📈 Kinetics & pKa

• k₁ ≤ 10⁴ s⁻¹ (based on buffer, pKa = 7)

• Actual hydration rate = 10⁶ s⁻¹ → buffer is essential

• Buffer effect: Rate = k’₁ × [Buffer]

✂ Restriction Enzymes – DNA Cleavage

Function:

• Found in bacteria to defend against viruses

• Cleave phosphodiester bonds in specific DNA sequences (recognition sites)

• Host DNA protected by methylation

🔬 Mechanism of Restriction Enzymes

DNA Cleavage Reaction:

• Involves in-line attack of 3′-oxygen on phosphorus

• Leaves a 5′ phosphoryl group

Mechanism Options:

1. Covalent intermediate (retains stereochemistry)

2. Direct hydrolysis (inverts stereochemistry) ✅ Correct

• Proven using sulfur substitution (phosphorothioates) → only one product forms

🧲 Role of Magnesium (Mg²⁺)

• Required for activity

• Mg²⁺:

  • Activates H₂O for nucleophilic attack

  • Coordinates with Asp residues in enzyme (e.g., Asp90, Asp74 in EcoRV)

  • Binds DNA to position scissile bond

🧬 Specificity & DNA Recognition

• Enzymes like EcoRV bind to cognate DNA with twofold symmetry

• Cognate DNA is distorted to allow catalysis

• Noncognate DNA isn’t distorted → no cleavage

Cognate Binding:

• Increases binding energy

• Drives conformational change → aligns phosphate with Mg²⁺

Host DNA Protection:

• Host methylases add CH₃ to bases in recognition sites

• Prevents restriction enzyme binding/distortion

🏋 Myosin – Mechanical Work via ATP Hydrolysis

Structure:

• Found in all eukaryotes

• 40+ genes in humans

• Has:

  • N-terminal ATPase domain (globular)

  • C-terminal coiled-coil tail

⚡ ATP Hydrolysis Mechanism

• ATP hydrolysis: Water attacks γ-phosphate

• Needs Mg²⁺ or Mn²⁺ to bind ATP → stabilizes phosphates

• Forms pentacoordinate transition state

• Modeled using vanadate (VO₄³⁻) analog

Steps:

1. ATP binds (with Mg²⁺)

2. Water (helped by Ser236) attacks γ-phosphate

3. Conformational change drives mechanical movement

🔄 Conformational Changes Drive Motion

• Structural change in ATPase domain → 25 Å shift

• Rate-limiting step: Pi release (not hydrolysis itself)

• Reaction is reversible (shown by isotope studies)

Visualization:

• Labeled myosin on actin + ATP → moves in 74 nm steps

• Model: “Walking” motion of myosin V along actin

🔁 Myosins Are P-loop NTPases

• Contain P-loop (phosphate-binding loop)

• Part of NMP kinase family (bind ATP, ADP)

• Myosin uses ATP energy for movement

⚙ ATP Synthase (Bonus)

• F₁ subunit: α₃β₃γδε arrangement

• α and β are P-loop proteins

• Only β subunits catalyze ATP synthesis

• Need full synthase + proton gradient to release ATP