Peptide Bonds & Structure

Peptide Bond Hydrolysis Mechanism

Summary

Peptide bond hydrolysis is a nucleophilic acyl substitution reaction where water attacks the carbonyl carbon, cleaving the C-N bond. While thermodynamically favorable, the reaction is kinetically slow due to the resonance stabilization of the peptide bond. Proteases accelerate this reaction by factors of 10⁹-10¹² through various catalytic strategies.

Key Points

  • 1Peptide bonds are thermodynamically unstable but kinetically stable (t½ ~400 years uncatalyzed)
  • 2Hydrolysis proceeds via nucleophilic attack on carbonyl carbon forming tetrahedral intermediate
  • 3Proteases achieve 10⁹-10¹² rate enhancement through diverse catalytic mechanisms
  • 4All proteases stabilize the tetrahedral transition state through oxyanion holes
  • 5Four major protease classes: serine, cysteine, aspartic, and metalloproteases

Peptide bond hydrolysis—the cleavage of the amide linkage with the addition of water—is fundamental to protein turnover, digestion, and countless cellular processes.

Thermodynamics vs Kinetics

The hydrolysis of peptide bonds is thermodynamically favorable (ΔG° ≈ -2 to -4 kcal/mol under physiological conditions), yet peptide bonds are remarkably stable:

- Half-life in water at pH 7: Approximately 350-600 years at 25°C

- Reason: The resonance stabilization of the peptide bond creates a high activation energy barrier (~20 kcal/mol)

This kinetic stability is essential—without it, proteins would spontaneously degrade too quickly for life to function.

The Uncatalyzed Mechanism

Spontaneous peptide bond hydrolysis proceeds via nucleophilic addition-elimination:

Step 1: Nucleophilic Attack

A water molecule (or hydroxide ion at high pH) attacks the electrophilic carbonyl carbon, forming a tetrahedral intermediate. The partial double-bond character of the C-N bond must be broken during this step.

Step 2: Tetrahedral Intermediate

The tetrahedral intermediate features:

  • A negatively charged oxygen (oxyanion)
  • The original carbonyl carbon now sp³ hybridized
  • Both the attacking oxygen and the leaving nitrogen attached

    • Step 3: Elimination

    The C-N bond breaks as electrons flow to the nitrogen, which becomes protonated to form an amine product. The other product is a carboxylic acid.

    Protease Catalytic Strategies

    Proteases have evolved multiple mechanisms to accelerate hydrolysis:

    Serine Proteases (Trypsin, Chymotrypsin)

    - Use a catalytic triad (Ser-His-Asp) for nucleophilic catalysis

  • The serine hydroxyl acts as a more potent nucleophile than water

    • - Form an acyl-enzyme intermediate that is subsequently hydrolyzed

  • Rate enhancement: ~10¹⁰-fold

    • Cysteine Proteases (Papain, Caspases)

  • Similar mechanism to serine proteases but use cysteine thiol as nucleophile
  • The thiolate anion (Cys-S⁻) is an even better nucleophile
  • Important in apoptosis and immune signaling

    • Aspartic Proteases (Pepsin, HIV Protease)

  • Use two aspartate residues to activate a water molecule

    • - General acid-base catalysis without covalent intermediate

  • One Asp acts as a base (activates water), the other as an acid (protonates leaving group)

    • Metalloproteases (Carboxypeptidase, MMPs)

  • Use a bound metal ion (usually Zn²⁺) to polarize the carbonyl
  • Metal-bound hydroxide is a potent nucleophile
  • The metal also stabilizes the tetrahedral intermediate

    • Transition State Stabilization

    All proteases share a common strategy: stabilizing the tetrahedral transition state. This is accomplished through:

    1. Oxyanion hole: Backbone NH groups or side chains donate hydrogen bonds to the developing negative charge on the carbonyl oxygen

    2. Substrate binding: Precise positioning of the scissile bond relative to catalytic machinery

    3. Electrostatic stabilization: Complementary charges in the active site

    pH Dependence

    Peptide hydrolysis is accelerated at both acidic and basic pH:

    - Acidic conditions: Protonation of carbonyl oxygen makes carbonyl more electrophilic

    - Basic conditions: Hydroxide is a better nucleophile than water

    - Proteases: Maintain optimal ionization states of catalytic residues within their pH optima

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