Chymotrypsin

Question 1

Which enzyme class is chymotrypsin part of? What does it do?

Answer 1

Hydrolases. It’s a non-specific serine protease that cleaves peptide bonds after a phenylalanine (hydrophobic residues)

Question 2

What are the 4 catalytic residues in chymotrypsin?

Answer 2

Asp 102, His 57, Ser 195, Gly 193

Question 3

Which 3 amino acids are part of the catalytic triad?

Answer 3

Asp, His, Ser

Question 4

What are the 3 catalytic strategies used by chymotrypsin?

Answer 4

Covalent catalysis, acid/base catalysis, and transition state stabilization

Question 5

Which part of the active site of chymotrypsin is involved in covalent catalysis?

Answer 5

Ser 195

Question 6

Which part of the active site of chymotrypsin is involved in acid/base catalysis?

Answer 6

His 57

Question 7

Which part of the active site of chymotrypsin is involved in transition state stabilization?

Answer 7

The oxyanion hole

Question 8

How is Ser 195 able to act as a nucleophile when a hydroxyl group is normally an extremely lousy nucleophile?

Answer 8

It is in the catalytic triad and is able to be deprotonated, which forms an alkoxide ion (which is a very good nucleophile)

Question 9

What is the general overview of the mechanism of chymotrypsin?

Answer 9

1. Substrate binds to active site
2. Nucleophilic attack by Ser 195
3. Collapse of the first tetrahedral intermediate and release of product 1
4. Hydrolysis of the acyl-enzyme intermediate
5. Hydrolysis continues
6. Collapse of second tetrahedral intermediate
7. Product 2 is released and the free enzyme is regenerated

Question 10

What is happening during step 1 of the chymotrypsin mechanism?

Answer 10

The side chain of the phenylalanine adjacent to the scissile peptide bond is positioned in the hydrophobic pocket. His 57 activates Ser 195 as a nucleophile by deprotonating it

Question 11

What is happening during step 2 of the chymotrypsin mechanism?

Answer 11

Ser 195 nucleophilicly attacks the carbonyl of the scissile bond and forms a covalent tetrahedral intermediate. The oxyanion of the carbonyl is stabilized in the oxyanion hole by H bonds from Gly 193 and Ser 195 backbones

Question 12

What is happening during step 3 of the chymotrypsin mechanism?

Answer 12

The amino of the scissile bond gets protonated by His 57 to make it a better leaving group. The tetrahedral intermediate collapses and the amino side of the scissile bond leaves as product 1. The other half stays covalently attached as an acyl-enzyme intermediate

Question 13

What is happening during step 4 of the chymotrypsin mechanism?

Answer 13

An incoming H2O gets deprotonated by His 57 and the hydroxide attacks the ester bond in the acyl-enzyme intermediate

Question 14

What is happening during step 5 of the chymotrypsin mechanism?

Answer 14

The attack on the ester bond generates the second tetrahedral intermediate. The oxyanion of the intermediate is stabilized in the oxyanion hole

Question 15

What is happening during step 6 of the chymotrypsin mechanism?

Answer 15

His 57 protonates Ser 195. The tetrahedral intermediate collapses and forms product 2, which is the carbonyl side of the scissile bond

Question 16

What is happening during step 7 of the chymotrypsin mechanism?

Answer 16

Product 2 is released and the free enzyme is regenerated

Question 17

How is His 57 able to deprotonate Ser 195 and H2O?

Answer 17

Asp 102 in the catalytic triad

Question 18

How is the transition state stabilized more than the substrate?

Answer 18

As an oxyanion transition state it forms 2 H bonds with the enzyme, and the substrate carboxyl only forms 1