Lecture 16 - Serine proteases

Question 1

What do all serine proteases have in common/what do they do?

Answer 1

have catalytic triad of Serine, Histidine, and Aspartate

All cleave peptide bond

Question 2

What is P1 and P2?

Answer 2

P1 is from the break to the C-terminal, P2 is from break to N-terminal

Question 3

What is the general mechanism of chymotrypsin

Answer 3

Enzyme binds with P2, releases P1 (fast) then releases P2 (slow)

Question 4

Describe the chymotrypsin mechanism

Answer 4

Step 1: alkoxide ion forms. His57 abstracts proton from Ser195 to produce alkoxide ion which is very strong nucleophile. Abstracting proton creates + on His57, which is stabilized by - on Asp102.
Step 2: bulky hydrophobic residue inserts into hydrophobic pocket, nucleophile attacks carbonyl carbon. This creates bond with Ser195, creates tetrahedral intermediate when carbonyl double bond broken. This is stabilized in the oxyanion hold. Collapse of the tetrahedral intermediate cleaves the peptide bond, which then abstracts the proton form His57 to create P1 and acyl-enzyme.
Step 3: water comes in, has one proton abstracted by His57 again, this creates oxygen nucleophile to attack carbonyl carbon, creates second tetrahedral intermediate which is stabilized in the oxyanion hole, again collapses and this time, the bond with Ser195 is cleaved, Ser195 then attacks and abstracts the proton back from His57.

Question 5

How is the oxyanion hole stabilized?

Answer 5

Tetrahedral intermediate stabilized by H-bonding with Ser195 and Gly193