Serine Proteases

Question 1

What proteases cleave serine

Answer 1

Trypsin, Chymotrypsin, Elastase

Question 2

Trypsin

Answer 2

Positively charged residue

Question 3

Chymotrypsin

Answer 3

Bulky hydrophobic residue

Question 4

Elastase

Answer 4

Small neutral residue

Question 5

Specificity Pocket

Answer 5

• This is how enzymes generate specificity for the enzymes that they want to cleave
• Adjacent to the active site of proteins

Question 6

Describe the specificity pockets of trypsin and chymotrypsin

Answer 6

Specificity pockets = deep

Question 7

Describe the specificity pocket of Elastase

Answer 7

Shallow

Question 8

Catalytic triad

Answer 8

• Asp
• His
• Ser

Question 9

Describe the catalytic strategy of serine protease

Answer 9

1. Serine is ideally placed to perform a nucleophilic attack on peptide bond (entropy reduction) BUT serine is NOT a good nucleophile
2. Histidine acts as a good base by extracting a proton from serine to make it a good nucleophile (general base catalysis) But histidine is NOT a good base
3. Aspartic acid side chain interacts with histidine side chain polarizing electrons in histidine making it a better base
   - Result is the formation of a covalent bond (tetrahedral intermediate (transition state)) (covalent catalysis)

Question 10

Describe the next steps in the catalytic strategy of serine proteases (how is the missile bond of the tetrahedral intermediate (transition state) broken)

Answer 10

• General acid catalysis breaks fissile bond of tetrahedral intermediate (histidine donates proton)
• Histidine makes water a good nucleophile by deprotonating it
• Water cleaves the remaining bond between the enzyme and substrate forming a new tetrahedral intermediate with a covalent bond (covalent catalysis)
• Histidine donates a proton to the tetrahedral intermediate to create a carboxylate product + the active enzyme (general acid catalysis)
  Enzyme is completely regenerated at the end

Question 11

Describe how serine proteases preferentially bind to the transition state?

Answer 11

• Transition state is stabilized by the formation of H-bonds with the tetrahedral intermediate in oxy anion hole
• Free energy and activation energy = lowered

Question 12

Zymogens

Answer 12

Inactive precursors to proteases

Question 13

How are zymogens converted to active proteases

Answer 13

Proteolytic cleavage
This is typically a cascading event