1. Lecture 13: Mechanism of Chymotrypsin
Chemical Mechanism
of Chymotrypsin

2. Chymotrypsin
Chymotrypsin is a digestive protease involved in breakdown of
proteins and peptides so that their amino acids can be used.
It is synthesized in the pancreas of mammals and released into
the digestive tract.
When first synthesized it is as a single polypeptide chain
in an inactive form, chymotrypsinogen, which must be activated
before the enzyme can fulfill its role.
Activation of chymotrypsin is achieved by “clips” in its polypeptide
chain, so active chymotrypsin consists of three distinct chains.
These remain bound together in a single domain, covalently held
together by disulfide bonds.
The activity of chymotrypsin is regulated by controlling when the
“clips” are made.

3. DG DG DG’ Acid + Amine Amide + H2O
Net Reaction:
Acid Amine
Amide + H2O
Overall DG of hydrolysis is negative (favorable).
Uncatalysed
pathway DG
uncat DG
cat
Reactants
DG’
Enzyme-catalysed
pathway
Products

4. Overview: E ES E-PN PC E-PN EPN S PN PC (Bond to be cleaved.) S PC PN
Substrate (S) binds.
Phase 1:
Enzyme creates nucleophile from serine side-chain.
Nucleophile attacks substrate.
Covalent intermediate is formed with second product ( PN )
bonded to serine, and first product ( PC ) is released.
Phase 2:
Enzyme creates a nucleophile from a water molecule.
Nucleophile attacks covalent intermediate, breaking covalent
bond to serine.
Second product ( PN ) is released. S PC PN E ES
E-PN PC
E-PN
EPN
Substrate
Binding
Chemical
Rearrangement
Product #1
Released
Chemical
Rearrangement
Product #2
Released

5. DG DG’ E+S ES ES E-PNPC E-PN EPN E+PN +PC EPN E-PNPC + PC + PC cat
Reactants
DG’ ES
E-PNPC
Products

6. Clues about mechanism:
Burst phase indicates a covalent intermediate is formed.
Kinetics experiments are used to figure out how
many steps there are in a reaction mechanism
and how long each step takes.
Chemical labeling with DIPF finds one particular serine residue (out of 28)
that is extremely reactive.
Chemical labeling experiments are used to figure
out which residues are responsible for important
steps in a reaction mechanism.
Crystal structure analysis reveals a catalytic triad, a group of 3 side-chains
which are responsible for the peculiar reactivity of this serine.
Determination of the crystal structure of an enzyme
provides a detailed description of the three-dimensional
arrangement of the molecule and in particular of the active
site.

7. (released immediately)
Chymotrypsin Kinetics
(remains covalently
bound)
(released immediately)
Very early in reaction,
p-nitrophenolate is
released giving rise to
the burst phase.
Subsequent reactants
must wait for an active
site to become available
through release of an
intermediate, giving
rise to the
steady-state phase.
Km = 20 mM
kcat = 77 s-1

8. Labelling of Serine 195 Inactivates Chymotrypsin
DIPF, an irreversible inhibitor, is a group-specific reagent for serine
residues. It forms a covalent adduct on Serine 195, which renders
the enzyme inactive. Only Ser 195, out of 28 serines in chymotrypsin,
is labelled, suggesting it is both especially reactive and that this
reactivity is necessary for catalysis.

9. Structure of Chymotrypsin
Globular single-domain protein.
Originally synthesized as a 245
residue protein, chymotrypsinogen.
Dipeptides and are
clipped out, tranforming the protein
into active chymotrypsin.
Therefore it has 3 chains (red, blue,
green) but these are covalently linked
by disulfide bridges.
The reactive serine 195 is located
in a cleft on the molecule, the
active site. Ser 195 is adjacent to
His 57 and Asp 102 which are
responsible for its reactivity.

10. Substrate (S) binds.
Enzyme creates nucleophile from serine side-chain.
Nucleophile attacks substrate.
Acyl-enzyme is formed with second product ( PN )
bonded to serine, and first product ( PC ) is released.
Enzyme creates a nucleophile from a water molecule.
Nucleophile attacks acyl linkage, breaking covalent
bond to serine.
Second product ( PN ) is released.
Phase 1:
Phase 2:

11. Substrate binding

12. Creation of Nucleophile
A nucleophile is a highly reactive, electron-rich group.
In chymotrypsin, serine 195 is converted into an alkoxide ion, a powerful nucleophile, through removal of its hydroxyl proton.
This difficult task is accomplished by the charge relay system between Asp 102, His 57, and Ser 195, which comprise the catalytic triad. His 57 can alternately accept or donate protons, while
stabilized by Asp 102.
(This is a good example of a general base in catalysis.)
The charges are stabilized by electrostatic effects.

13. Nucleophilic Attack
The carbonyl carbon on the substrate has 3 bonds and so is a trigonal atom.
The alkoxide ion attacks the carbonyl carbon, forming a tetrahedral
intermediate with 4 bond to that carbon.
The former carbonyl oxygen is converted into a negatively charged group,
the oxyanion, which is stabilized by by an arrangement of partial positive
charges nearby in the oxyanion hole. (an electrostatic effect)

14. Formation of Acyl-enzyme
The tetrahedral intermediate breaks down when the histidine donates a
proton and creates a new amino group on the terminus of the first
product ( PC ), which is released. (His 57 is acting as general acid.)
The remainder of the substrate remains attached to the enzyme through
an ester linkage to Serine 195. (Covalent catalysis.)
Acyl group

15. Creation and Use of New Nucleophile
Another nucleophile is created by the enzyme, using His 57 to withdraw
a proton from a water molecule to form a hydroxide ion. (another example
of general base catalysis)
This nucleophile attacks the acyl carbon forming a second tetrahedral
intermediate, which is again stabilized by the oxyanion hole. (Another
example of the electrostatic effect.)

16. De-acylation Step
The tetrahedral intermediate breaks down when His 57 donates a proton
to serine 195, displacing the acyl group and regenerating the serine
hydroxyl group. The second product ( PN ) is released, concluding
the reaction.

18. Subtilisin Carboxypeptidase A Same Chemistry, Different Enzymes
Other unrelated classes of proteases, whose sequences and structures
are unrelated to those of chymotrypsin, nevertheless have the same spatial arrangement of the His-Asp-Ser catalytic triad.
The same catalytic method appears to have arisen independently at least three times in nature- an example of convergent evolution.
Subtilisin
Carboxypeptidase A

19. Summary:
Chymotrypsin is a protease and its activity is regulated by controlled
cleavage of its backbone.
Its chemical mechanism proceeds in two stages:
1. Nucleophilic attack on substrate by Ser 195 to form acyl-enzyme
complex
followed by
2. Deacylation though nucleophilic attack by water on the acyl intermediate.
Key Concepts:
Meaning of burst phase and labelling of Serine 195
Catalytic triad: Roles of His 57, Asp 102, and Ser 195 in mechanism
Occurrences of acid-base catalysis and covalent catalysis in mechanism