Serine Proteases Overview

Serine Proteases

• Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, and tissue plasminogen activator are all serine proteases.
• They are secreted as proenzymes or zymogens.
• Serine proteases cleave polypeptide chains.
• They have similar sequences, structures, and mechanisms.
• Acetylcholinesterase also has a similar mechanism to serine proteases, but is not a protease.

Trypsin, Chymotrypsin and Elastase

• Trypsin cleaves after Arg or Lys (basic amino acids) except when either is followed by Pro.
• Chymotrypsin cleaves after Phe or Tyr (aromatic amino acids).
• Elastase cleaves after small, neutral amino acid residues.

Structure

• Chymotrypsin (and other serine proteases) contains a catalytic triad, which is composed of Asp, His, and Ser.

Substrate Binding Pocket

• The nature of the pocket determines the specificity of the enzyme.
• Trypsin has a negatively charged substrate-binding pocket for basic residues.
• Chymotrypsin has a hydrophobic pocket to bind aromatic residues.
• Elastase has a bulky substrate-binding pocket with Thr and Val residues to accommodate small amino acids.

Chymotrypsin Kinetics

• Chymotrypsin kinetics exhibit burst kinetics- this suggests a fast initial step and a slow second step.
• Burst kinetics are assayed using an artificial substrate that releases a product that absorbs at 400 nm.

Serine Protease Mechanism

• Serine proteases use a combination of covalent and general acid-base catalysis.
• The catalytic triad works as follows:
  • Asp orients and stabilizes His.
  • His acts as a general acid and base.
  • Ser195 forms a covalent bond with the peptide to be cleaved, changing the trigonal carbon to tetrahedral.
  • The tetrahedral oxyanion intermediate is stabilized by Gly193 and Ser195.
  • The oxyanion hole is composed of two amide groups.

Catalytic Triads

• Found in several hydrolases and transferase enzymes.
• Involve divergent and convergent evolution
  • Residues are far away in the primary structure.
• Catalytic triads are composed of:
  • Acid that orients and stabilizes the base (Asp, Glu, His)
  • Base that polarizes the nucleophile (His, rarely Lys)
  • Nucleophile that attacks the substrate (Ser, Cys, sometimes Thr)

Aspartic Proteases

• Aspartic proteases have a different structure and mechanism than serine proteases.
• They contain two Asp residues in the active site.
• They cleave peptide bonds between two hydrophobic amino acids.
• They do not involve covalent catalysis.

pH Dependence of Aspartic Proteases

• Aspartic proteases are active at acidic pH.
• The mechanism requires that one Asp is protonated and the other is deprotonated when the substrate binds.
• The LBHB allows 'hydrogen tunneling'.

HIV-1 Protease

• A novel aspartic protease, HIV-1 protease, cleaves polyprotein products of the HIV genome.
• It is also a remarkably good imitation of mammalian aspartic proteases.
• HIV-1 protease is a homodimer, which makes it more genetically economical for the virus.
• It has a two-fold symmetric active site with different 'flaps'.

Protease Inhibitors for AIDS Patients

• Protease inhibitors are a common treatment for AIDS.
• They block the active site of HIV-1 protease so new viral particles cannot be created.
• Structure-based drug design has been used to create several inhibitors that work in a dish.