Catalytic Strategies 1

⚙ General Properties of Enzymes

Enzymes are biological catalysts that dramatically increase the rate of reactions under biological conditions.

🔑 Key Features:

• Higher Reaction Rates (catalytic power)

• Milder Reaction Conditions (neutral pH, moderate temperature, etc.)

• Greater Reaction Specificity (react with specific substrates)

• Capacity for Regulation (can be activated or inhibited)

🔬 Fundamental Catalytic Strategies

Enzymes use a few core catalytic principles—often in combination—to achieve their effects.

1. Covalent Catalysis

• The active site contains a nucleophilic group that forms a transient covalent bond with the substrate.

• Example: Chymotrypsin uses Serine-195 as a reactive nucleophile.

2. Acid-Base Catalysis

• A molecule other than water donates or accepts a proton to facilitate the reaction.

• Example: Histidine in chymotrypsin acts as a base; carbonic anhydrase and myosin also use this strategy.

3. Catalysis by Approximation (Proximity and Orientation Effects)

• The enzyme brings two substrates close together in the correct orientation.

• Example: Carbonic anhydrase

4. Metal Ion Catalysis

• Metal ions can:

  • Act as electrophilic catalysts

  • Stabilize negative charges

  • Generate nucleophilic OH⁻

• Common metals: Zn²⁺, Fe²⁺, Mg²⁺

🔗 Binding Energy in Enzyme-Substrate Interactions

🔬 What is Binding Energy?

• Free energy released when a substrate binds to the enzyme via multiple weak, specific interactions.

🔄 Functions of Binding Energy:

• Substrate specificity

• Catalytic efficiency

• Stabilizes the transition state

• Induces conformational changes (induced fit)

📉 Enzymes Lower Activation Energy

Key Concepts:

• Y-axis: Energy

• X-axis: Reaction progress from substrate to product

• Activation energy = Energy difference between substrate and transition state

⚙ Role of Enzymes:

• Enzymes lower the activation energy barrier, increasing the rate of reaction without changing the overall ΔG.

🔪 Proteases and Chymotrypsin

🧪 General Function:

• Proteases cleave peptide bonds through hydrolysis.

• Although exergonic, peptide bond hydrolysis is kinetically slow due to resonance stabilization.

🧬 Chymotrypsin: Overview

• A serine protease secreted by the pancreas.

• Cleaves peptide bonds after large hydrophobic residues (e.g., Phe, Trp, Tyr, Met).

• Uses a catalytic triad: Ser195, His57, Asp102

🔍 Mechanism of Chymotrypsin Catalysis

1. Highly Reactive Ser195

• Ser195 becomes a powerful nucleophile during catalysis.

• DIPF irreversibly modifies only Ser195, confirming its role.

2. Reaction Occurs in Two Steps:

• Step 1: Acylation – Ser195 attacks peptide bond → forms acyl-enzyme intermediate

• Step 2: Deacylation – Water hydrolyzes the intermediate → regenerates free enzyme

3. Chromogenic Substrates for Study

• Substrate: N-Acetyl-L-phenylalanine-p-nitrophenyl ester

• Product: p-Nitrophenolate (yellow, measurable by spectrophotometry)

📈 Kinetics of Chymotrypsin

• Two phases detected using stopped-flow spectroscopy:

  • Fast pre-steady state: Acylation

  • Slower steady state: Deacylation

🔺 The Catalytic Triad

• Forms a hydrogen-bond network critical for catalysis.

• Common to many serine proteases.

🎯 The Specificity Pocket

• S1 pocket: Deep, hydrophobic

• Binds P1 residue of substrate (large hydrophobic side chains)

• Aligns scissile bond for cleavage

• Specificity determined by enzyme-substrate residue matching (S1-P1, S2-P2, etc.)

🌍 Evolutionary and Structural Insights

🧬 Catalytic Triads Across Enzymes

• Found in many hydrolytic enzymes

• Present in enzymes not homologous to chymotrypsin

• Evidence for convergent evolution

🔬 Site-Directed Mutagenesis of Subtilisin

• Each catalytic triad residue mutated to alanine

• Dramatic loss of activity, minimal change in K_M

• Suggests transition state stabilization still contributes to catalysis

🔁 Other Classes of Proteases

Not all proteases use Ser195. Other classes use different strategies to generate a nucleophile:

1. Cysteine Proteases

• Use histidine-activated cysteine

• Evolved independently multiple times

2. Aspartyl Proteases

• Use aspartate-activated water molecule

• Example: Renin, with twofold symmetry in structure

3. Metalloproteases

• Use a metal-activated water molecule

• Often include a base (like glutamate) to deprotonate water

🧬 Activation Strategies Summary

💊 Protease Inhibitors as Drugs

HIV Protease:

• A dimeric aspartyl protease

• Essential for maturation of the HIV virion

Indinavir (Crixivan):

• A substrate analog inhibitor

• Binds to active site, blocks maturation of virus

• Used in AIDS treatmentto reduce viral load and improve immune function in HIV-infected individuals.