CHYMOTRYPSIN

The biological role of chymotrypsin is to catalyze the hydrolysis of proteins in the small
intestine. Chymotrypsin does not cleave all peptide bonds at a significant rate. Instead,
it is selective for peptide bonds on the carboxyl side of the aromatic side chains of
tyrosine, tryptophan and phenylalanine and of large hydrophobic residues such
as methionine. Chymotrypsin also hydrolyzes ester bonds in which case it selects for a
hydrophobic acyl group. Chymotrypsin belongs to a family of proteins called the serine
proteases. Trypsin and thrombin belong to this important family.

Chymotrypsin is a 25 kd protein composed of three polypeptide chains joined by two

interchain disulfide bonds.

Chymotrypsin folds compactly with all charged residues on the surface with the
exception of the residues in the active site of the enzyme. Chymotrypsin contains
several regions of b conformation, but little a helix.

Chymotrypsin catalyzes the hydrolysis of peptide or ester bonds in two distinct stages.

1. ACYLATION. In this stage ser 195 of the enzyme reacts with the carbonyl group of
the peptide bond being attacked to form an acyl-enzyme intermediate. This releases the
amine component of the substrate.

acyl enzyme amine

component of
intermediate
the substrate
Ser 195 substrate O
CH2 OH + O
CH2 O C R' + RNH 2
C R'

N H

2. DEACYLATION. In this stage the acyl-enzyme intermediate is hydrolyzed by water.

This releases the acid component of the substrate.
acid component
Ser 195 of the substrate
enzyme
O O
CH 2 O C R' + H2 O CH 2 OH + R' C OH
HOW DOES THE ENZYME CHYMOTRYPSIN MAKE THE ABOVE TWO STAGE
PROCESS POSSIBLE?

The catalytic activity of chymotrypsin depends on the unusual reactivity of serine 195. A
-CH2OH is usually quite unreactive under physiological conditions. What makes it so
reactive in the active site of chymotrypsin? The operation of a catalytic triad makes
serine 195 a more powerful nucleophile. This catalytic triad is composed of serine 195,
aspartic acid 102 and histidine 57.

O CH
Asp C O - ......... HN N ........ HO Ser 195
102
CH C
His 57

O CH
+ -
Asp C O ......... HN
- N H ........O Ser 195
102
CH C
His 57

When a substrate is not present, his 57 is in the non-protonated form. After the
substrate binds, his 57 accepts a proton from the hydroxyl group of serine 195 when the
oxygen atom of serine 195 initiates a nucleophilic attack on the substrate. Aspartate 102
remains negatively charged and functions to stabilize the positive charge on his 57 and
keeps his 57 optimally positioned to participate in the catalytic triad.

Histidine 57 and serine 195 participate directly in the cleavage of the susceptible peptide
bond of the substrate. Peptide bond hydrolysis starts with an attack of the oxygen atom
of the hydroxyl group of serine 195 on the carbonyl carbon atom of the susceptible
peptide bond. A transient tetrahedral intermediate is formed. This reaction is
facilitated by the catalytic triad described above, which serves to draw a proton away
from the nucleophile. Histidine 57 then donates a proton to the nitrogen atom of the
susceptible peptide bond. As a result, the susceptible peptide bond is cleaved. At this
stage, the amine component is hydrogen bonded to histidine 57 and the acid component
will quickly diffuse away leaving the acid component esterified to serine 195, the acyl-
enzyme intermediate. This completes the acylation stage of the reaction.
(diagram on next page)
 tetrahedral acyl enzyme
R' transition state intermediate
Ser 195 -O
R'
CH 2 OH O C C O
N CH 2 O N H CH 2 O C R'
R N H R
HC CH substrate H+
H amine
N C R N
H
N H component
CH 2 HC CH
N CH
N C
O- His 57
H HC C
CH 2 N
C O H CH 2
O- His 57
O-
Asp 102 C O
C O
Asp 102
Asp 102
The next stage is deacylation. The amine component of the substrate diffuses away,
and a water molecule takes its place at the active site. First, the catalytic triad draws a
proton away from water. The resulting OH- ion simultaneously attacks the carbonyl
carbon atom of the acyl group that is attached to serine 195. As in acylation, a transient
tetrahedral intermediate is formed. Histidine 57 then donates a proton to the oxygen
atom of serine 195, which results in the release of the acid component of the substrate.
The acid component of the substrate diffuses away and the enzyme is ready for another
round of catalysis.

acyl enzyme
intermediate
O tetrahedral acid
transition state component
CH 2 O C R' -O
R' O
H C
O O C
HO R'
H CH 2 O O H
N
HC CH H
N C N
H CH CH 2 O H
HC
CH 2
N C N CH
H
O- His 57 CH 2 HC C
N
C O O- His 57 H CH 2
C O O-
Asp 102 C O
Asp 102
Asp 102
NOTE: Chymotrypsin reduces the energy barrier by providing an alternative pathway for
the reaction through intermediate products of lowered internal energy, free energy. The
tetrahedral intermediates and the acyl-enzyme represent transition states. The free
energy of activation required to reach these transition states is lowered because of the
covalent and non-covalent interactions of chymotrypsin and its polypeptide substrate.

NO enzyme

internal enzyme
energy catalyzed

reactant
products

reaction progress