Chymotrypsin

Question 1

What are the classes of enzymes?

Answer 1

1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerase
6. Ligases
7. Translocases

Question 2

Oxidoreductases

Answer 2

Transfer e- between molecules (redox rxn)

Question 3

Transferases

Answer 3

Transfer functional groups between molecules

Question 4

Hydrolases

Answer 4

Cleave molecules by addition of water

Question 5

Lyases

Answer 5

Add atoms or functional groups to double bonds or remove them to form double bonds

Question 6

Isomerase

Answer 6

Move functional group within a molecule

Question 7

Ligases

Answer 7

Join two molecule together (powered by ATP hydrolysis)

Question 8

Translocases

Answer 8

Catalyze movement of ions or molecules across membranes or their separation within membranes

Question 9

What is chymotrypsin?

Answer 9

A proteolytic enzyme that ensures protein turnover (cleave peptide bonds through addition of H2O)
Chymotrypsin helps hydrolyze dietary proteins into small peptides and amino acids for absorption by the small intestine

Question 10

What is the mechanism of chymotrypsin?

Answer 10

Cleaves preferentially on the carboxyl side of large hydrophobic amino acids (F, W, I, M, Y)

Question 11

Chymotrypsin uses a catalytic triad

Answer 11

• His 57 accepts a H+ from Ser 195 hydroxyl group, creating a powerful nucleophile (alkoxide ion), uses general acid/base catalysis
• Asp 102 orients His 57 to act as a proton acceptor

Question 12

Chromogenic substrates can be used to monitor chymotrypsin activity

Answer 12

Yellow colour produced as chymo cleaves substrate = study rxn as it proceeds using spectrometer
Hydrolysis proceeds in two steps:
1. Burst phase
2. Steady-state phase (follows M-M kinetics)

Question 13

Chymotrypsin forms an acyl-enzyme intermediate

Answer 13

The two steps of hydrolysis can be explained by formation of an acyl-enzyme intermediate

• Taking H+ off Ser 195 is v fast
• But reforming the enzyme is a lot slower
• Formation of the acyl-enzyme intermediate uses covalent catalysis

Question 14

Peptide hydrolysis by chymotrypsin

Answer 14

Step 1: substrate binding
Step 2: Serine nucleophilic attack on peptide carbonyl group after N accepts its proton. Produces tetrahedral intermediate which is stabilized by oxyanion hole.
Step 3: Collapse of the tetrahedral intermediate
Step 4: Release of the amine component
Step 5: Water binds
Step 6: Water’s nucleophilic attack on the acyl-enzyme intermediate
Step 7: collapse of the tetrahedral intermediate
Step 8: Release of the carboxylic acid component

Question 15

Summary of the reaction mechanism:

Answer 15

Double displacement (2 subs and 2 prods released)

• Specificity pocket for bulky hydrophobic side chains of amino acids
• Chymotrypsin is a hydrolase