Biochem Chymotrypsin Mechanism (Final)

Step 1 of Chymotrypsin Mechanism

substrate binds to the enzyme

substrate has an aromatic side chain that rests in the specificity pocket, this places the bond to be cleaved near the catalytic serine, chymotrypsin cleaves the peptide bonds directly following (on the C-terminal side of) aromatic amino acids

Step 2 of Chymotrypsin Mechanism

base catalysis by His 57 in the catalytic triad

deprotonates the catalytic serine (195) to create the strong nucleophile

Step 3 of Chymotrypsin Mechanism

negatively charged oxygen of serine (nucleophile) attacks the C of the substrate peptide bond

nucleophilic attack step, electrons move to the oxygen of the substrate, creating a tetrahedral oxygen

Step 4 of Chymotrypsin Mechanism

oxyanion is stabilized by the partial positive charges in the oxyanion hole by interacting with the backbone amide hydrogens to form two H-bonds

this is a key step because it stabilizes the transition state

Step 5 of Chymotrypsin Mechanism

release of the first product- the C-terminal end of the original protein

proton from the His is transferred to the NH of the new product (new N-terminus), and the C-terminal half of the protein is released while the N-terminal half of the protein is still covalently attached to the enzyme

Step 6 of Chymotrypsin Mechanism

formation of second oxyanion intermediate

water enters the active site and is deprotonated by His to produce OH-, OH- attacks the C of the substrate, electrons move to the oxygen of the substrate which creates another oxyanion that is stabilized by the oxyanion hole

Step 7 of Chymotrypsin Mechanism

release of the second product- the N-terminal half of the substrate

enzyme is the leaving group in this case (ser detaches), serine is regenerated by transferring a proton from His

Step 8 of Chymotrypsin Mechanism

the catalytic triad is regenerated

active site is reset to catalyze again

Step 1 of Chymotrypsin Mechanism Diagram

Chymotrypsin Diagram

Step 2 of Chymotrypsin Mechanism Diagram

Step 3 of Chymotrypsin Mechanism Diagram

Step 4 of Chymotrypsin Mechanism Diagram

Step 5 of Chymotrypsin Mechanism Diagram

Step 6 of Chymotrypsin Mechanism Diagram

Step 7 of Chymotrypsin Mechanism Diagram

Step 8 of Chymotrypsin Mechanism Diagram

What is the first product released by chymotrypsin?

the c-terminal product (that has a new n-terminus)

First Chymotrypsin Product Diagram

What is left on the enzyme after the first product (c-terminal end) is released?

acyl-enzyme intermediate, n-terminal half is still attached to the enzyme

N-Terminal Attachment to Enzyme After C-Terminal Release Diagram