Chymotrypsin (detailed)

Question 1

Proteases cleave proteins by hydrolysis

Answer 1

H2O + peptide bond, hence it is favourable reaction.
But the reaction occurs very slowly, this is due to charge delocalisation. Hence proteins become less hydrlysable.
The e- on the CO double bond is transfered to CN.

Question 2

Chymotrypsin

Answer 2

Cleaves large hydrophobic amino acids eg Trp,Tyr,Phe,Met.

Question 3

Chymotrypsin covalent catalysis

Answer 3

Uses a powerful nucleophile to attack.

Nucleophiles can be C-O(-ve) or a lone pair of electrons.

Question 4

Chymotrypsine serine residue

Answer 4

Serine binds to F, inhibiting the enzyme.

Question 5

Production of p-nitrophenolate

Answer 5

2 step reaction
Burst phase and steady state.
Burst step is the absorption where the subtrate is taken in.
Slow step is waiting for the release of the substrate to get the free form enzyme.

Question 6

Catalytic triad

Answer 6

Serine 195, His57(imidazole ring), Asp102.
These amino acids are H-bonded to stabilize the structure.

In the presence of substrate, imidazole takes the proton of Serine (from H), this forms a reactive alkoxide ion (potent nucleophile).
Asp102 has a stabilizing role.

Question 7

Chymotrypsin reaction mechanism

Answer 7

The serine reside attacks central C atom, forming tetrahedral intermediate, this is unstable. the other part of the enzyme forms an oxyanion hole which stabilizes the O -ve on the carbon.

Question 8

Chymotrypsin and trypsin

Answer 8

They are very similar in structure, yet their peptidase specificities are different.

Because Trypsin has an Asp in its pocket, so can’t accomodate a large residue.