Exam 2: BIOC 3022- Lecture 16

Chymotrypsin

Digestive protease of the duodenum (proteolysis)

Serine protease

Active site serine covalently attaches to the substrate

What does chymotrypsin do?

Cuts peptide bonds after large nonpolar

residues and made as an inactive precursor,

chymotrypsinogen

Catalytic Triad

Ser, His, Asp. Ser is the residue used in covalent catalysis.

What is the function of the S1 pocket in chymotrypsin?

The hydrophobic pocket in the active site accommodates large, nonpolar residues.

What does chymotrypsin prefer to cut next to?

Large nonpolar residues

Trypsin pocket

Aspartic Acid (Asp), negatively charged pocket

Chymotrypsin pocket

Broad, nonpolar

Elastase pocket

Valine (Val), narrow, nonpolar

What role does Ser195 play in the Chymotrypsin mechanism?

The nucleophile (covalent catalysis)

What role does His57 play in the Chymotrypsin mechanism?

General base, then general acid (acid-base catalysis)

What role does Asp102 play in the Chymotrypsin mechanism?

Orients His57 and favors its proton abstraction

What role does the Oxyanion hole play in the Chymotrypsin mechanism?

Stabilizes the negative charge on the tetrahedral intermediate (electrostatic catalysis)

What is the first step to the Chymotrypsin mechanism?

Nonpolar side chain fits the hydrophobic S1 pocket, which positions the peptide bond for attack.

What is the second step to the Chymotrypsin mechanism?

His57 takes a proton from Ser195 (acid-base). Ser195 attacks the carbonyl carbon (covalent). Oxyanion hole stabilizes the negative oxygen (electrostatic).

What is the third step to the Chymotrypsin mechanism?

Protonated His57 donates a proton to the leaving amino group (acid-base). An acyl-enzyme intermediate forms (covalent).

What is the fourth step to the Chymotrypsin mechanism?

Product 1 has left and water takes its place. His57 acts as a general base on the water, making hydroxide (acid-base catalysis).

What is the fifth step to the Chymotrypsin mechanism?

Hydroxide attacks the acyl-enzyme carbonyl (covalent). Oxyanion hole stabilizes the second tetrahedral intermediate (electrostatic).

What is the sixth step to the Chymotrypsin mechanism?

Intermediate collapses and a carboxylate forms. His 57 donates a proton to Ser195, which leaves the substrate (acid-base catalysis).

What is the seventh step to the Chymotrypsin mechanism?

Product 2 dissociates. The free enzyme is ready for another substrate (catalyst regenerated).

Fast phase

Steps 1 to 3 (acyl-enzyme forms, product 1 leaves)

Slower phase

Steps 4 to 7 (water attacks, product 2 leaves)

Burst phase

Product 1 appears quickly, then the rate slows to the steady state.

Name the nucleophile and the general base in the chymotrypsin mechanism.

The nucleophile is Serine 195 (Ser195) and the general base is Histidine 57 (His57).

Why does chymotrypsin cut next to Phe, Tyr, or Trp?

Chymotrypsin cuts peptide bonds on the carboxyl (C-terminal) side of aromatic and bulky hydrophobic amino acids like phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp)

What is the covalent intermediate called, and which steps form and break

it?

The acyl enzyme intermediate formed in step 1 and broken in step 2.

Which catalytic strategy does the oxyanion hole illustrate, and what does it

stabilize?

The oxyanion hole illustrates transition-state stabilization. It stabilizes the negatively charged tetrahedral intermediate

Why is chymotrypsin made as chymotrypsinogen?

To prevent the enzyme from digesting and destroying the pancreatic cells and tissues where it is synthesized