Enzymes and Active Site Mechanisms: Chymotrypsin

Active Site Participants

  • The active site of an enzyme involves participants critical for both binding and catalysis.

Chymotrypsin Mechanism

  • Chymotrypsin is an enzyme that cleaves peptide bonds, specifically at the carboxyl side of tyrosine (Y), tryptophan (W), phenylalanine (F), and sometimes leucine.

  • The reaction involves the breaking of a C-N bond in a peptide using water (hydrolysis), resulting in two peptide fragments.

Key Components

  • S1 Pocket: A hydrophobic pocket in chymotrypsin that binds to hydrophobic residues of the substrate (P1).

  • His57 (Histidine 57): Acts as a general base catalyst.

  • Asp102 (Aspartic Acid 102): Works in conjunction with His57 to facilitate catalysis.

Stage I: Acylation

  1. Substrate Binding:

    • The substrate (peptide) binds to the active site, with the residue adjacent to the scissile bond (P1) fitting into the S1 hydrophobic pocket.

  2. General Base Catalysis:

    • His57 acts as a general base, abstracting a proton from a water molecule, which then performs a nucleophilic attack on the carbonyl carbon of the peptide bond.

    • Asp102 helps to orient and stabilize His57.

  3. Transition State #1:

    • A tetrahedral intermediate forms, stabilized by the active site.

    • Formation of a transition state with a developing oxyanion hole (δ−\delta^-) and a partial positive charge (δ+\delta^+).

  4. Peptide bond cleavage:

    • The C-N bond is broken, leading to the release of the first peptide fragment (P2) and the formation of an acyl-enzyme intermediate.

Intermediate State

  • Acyl-enzyme intermediate: P1 remains covalently attached to the enzyme (specifically to Serine).

Stage II: Deacylation

  1. Water Binding:

    • A water molecule binds to the active site.

  2. General Base Catalysis:

    • His57 acts as a general base, activating the water molecule for nucleophilic attack on the carbonyl carbon of the acyl-enzyme intermediate.

  3. Transition State #2:

    • Another tetrahedral intermediate forms.

    • Formation of a second transition state with a developing oxyanion hole (δ−\delta^-) and a partial positive charge (δ+\delta^+).

  4. Product Release:

    • The acyl-enzyme bond is broken, releasing the second peptide fragment and regenerating the free enzyme.

Regeneration

  • The enzyme is now ready to catalyze another reaction.