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:
Zn²⁺ reduces water's pKa from 15.7 to 7 → generates OH⁻
CO₂ binds next to Zn²⁺
OH⁻ attacks CO₂ → forms HCO₃⁻
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:
Covalent intermediate (retains stereochemistry)
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:
ATP binds (with Mg²⁺)
Water (helped by Ser236) attacks γ-phosphate
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