Chymotrypsin Mechanism

Question 1

Step 1: Nucleophilic Attack

Answer 1

• His acts as a general base by removing H+ from Ser-195
• Ser-195 becomes a better nucleophile as a result, and attacks the C=O bond in the peptide
• Neg charge on Asp-102 delocalizes the now positive charge on His-57

Question 2

What does it mean to delocalize a charge

Answer 2

stabilizing force by spreading energy over a large area

Question 3

Formation of the first transition state

Answer 3

• the oxyanion hole pulls O- into the transition state
• favours tetrahedral carboxyanion configuration

Question 4

Breakdown of the first transition state

Answer 4

• NH group acts as the leaving group
• His 57 acts as a general acid now and donates the H+ back to the leaving group

Question 5

Formation of acyl-enzyme intermediate

Answer 5

C-terminal peptide leaves
N-terminal peptide remains covalently bound
- intermediate in reaction

Question 6

Step 2: Nucleophilic Attack

Answer 6

• water enters catalytic site
• His-57 acts as general base and takes H+ from water
• water becomes a better nucleophile (has a new lone pair)

Question 7

Formation of the second transition state

Answer 7

oxyanion hole stabilizes the transition state

Question 8

Breakdown of the second transition state

Answer 8

• His-57 now acts as a general acid donating H+ to Ser-195
• breaks the acyl-enzyme bond

Question 9

Formation of the product

Answer 9

• N-terminal peptide leaves
• catalytic triad is regenerated
• ready to cycle again