CH 8

8.1 Basic Catalytic Strategies

• Common catalytic strategies used by many enzymes:
  1. Covalent catalysis: Active site nucleophile is briefly covalently modified.
     • Example: Serine 195 in chymotrypsin.
  2. General acid-base catalysis: Molecule other than water donates or accepts a proton.
     • Example: Histidine 57 in chymotrypsin.
  3. Metal ion catalysis: Metal ions act as electrophilic catalysts.
  4. Catalysis by approximation and orientation: Enzyme brings two substrates together in an orientation that facilitates catalysis.

8.2 Modulation of Enzyme Activity

• Enzyme activity is modulated by:
  • Temperature
    • Example: tyrosinase
  • pH
    • Enzymes have an optimal pH.
• Reversible Inhibition:
  1. Competitive inhibition: Inhibitor is structurally similar to the substrate and binds to the active site.
     • V_{max} is unchanged.
     • K_M increases.
     • Can be overcome by high substrate concentration.
  2. Uncompetitive inhibition: Inhibitor binds only to the enzyme-substrate complex.
     • V_{max} is lower.
     • K_M is lower.
     • Cannot be overcome by excess substrate.
  3. Noncompetitive inhibition: Inhibitor binds to either the enzyme or enzyme-substrate complex.
     • V_{max} is lower.
     • K_M is unchanged.
     • Cannot be overcome by increasing substrate concentration.

Lineweaver-Burk Plots

• Lineweaver-Burk plots illustrate differences in reversible inhibition:
  • Competitive: Intersect on the y-axis (same V{max}, different KM).
  • Uncompetitive: Parallel lines (both V{max} and KM are affected).
  • Noncompetitive: Intersect on the x-axis (same KM, different V{max}).

Irreversible Inhibitors

• Irreversible inhibitors bind tightly, often covalently, to enzymes.
• Used to map the active site.
• Types:
  • Group-specific reagents: React with R groups of specific amino acids.
    • Example: Diisopropylphosphofluoridate (DIPF) for serine proteases.
  • Affinity labels (substrate analogs): Structurally similar to substrate, covalently modify active site amino acids.
    • Example: Tosyl-L-phenylalanine chloromethyl ketone (TPCK).
  • Mechanism-based (suicide inhibitors): Enzyme participates in its own inactivation.
    • Examples: Penicillin, phenylmethanesulfonyl fluoride (PMSF).
  • Transition state analogs: Mimic the transition state.
    • Example: Allopurinol.

8.3 Chymotrypsin: Catalysis and Inhibition

• Chymotrypsin: Proteolytic enzyme that hydrolyzes peptide bonds on the carboxyl side of large hydrophobic amino acids.
• Serine 195 acts as a nucleophile attacking the carbonyl group of the protein substrate.
  • DIPF modifies Serine 195, inhibiting the enzyme.

Chymotrypsin Action

• Proceeds in two steps linked by a covalently bound intermediate:
  • Acylation: Rapid formation of an acyl-enzyme intermediate.
  • Deacylation: Slower release of the acyl group, regenerating the free enzyme.
• N-acetyl-L-phenylalanine p-nitrophenyl ester is a chromogenic substrate for chymotrypsin, generating colored products for enzymatic studies.
• Chromogenic substrate studies reveal two stages:
  • Rapid pre-steady state
  • Slower steady state

Catalytic Role of Histidine 57

• Affinity label TPCK covalently modifies Histidine 57, leading to loss of enzyme activity.

Catalytic Triad

• Serine 195, Histidine 57, and Aspartic Acid 102 form a catalytic triad.
  • Histidine 57 removes a proton from Serine 195, generating a reactive alkoxide ion.
  • Aspartic acid 102 orients histidine and makes it a better proton acceptor.

Oxyanion Hole

• Oxyanion hole stabilizes the tetrahedral reaction intermediate.
  • Hydrogen bonds from NH groups stabilize the charged oxygen on the intermediate.

S1 Pocket

• The specificity of chymotrypsin is determined by the S1 pocket, a crevice that binds to a residue on the substrate and positions the adjacent peptide bond for cleavage.