Lecture 13: Mechanism of Chymotrypsin

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Lecture 13Mechanism of Chymotrypsin

• Chemical Mechanism
• of Chymotrypsin

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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.
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Net Reaction
Overall DG of hydrolysis is negative (favorable).
Uncatalysed pathway
cat
Reactants
DG
Enzyme-catalysed pathway
Products
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Overview
S
PN
PC
(Bond to be cleaved.)
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.
PN
PC
S
E
ES
EPN
E-PN PC
E-PN
Substrate Binding
Chemical Rearrangement
Product 1 Released
Chemical Rearrangement
Product 2 Released
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ES
ES
E-PNPC
E-PN PC
EPN PC
EPN PC
cat
EPN
Reactants
DG
ES
E-PNPC
Products
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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. Determinatio
n of the crystal structure of an enzyme provides
a detailed description of the three-dimensional a
rrangement of the molecule and in particular of
the active site.
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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
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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.
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Structure of Chymotrypsin
Globular single-domain protein. Originally
synthesized as a 245 residue protein,
chymotrypsinogen. Dipeptides 14-15 and 147-148
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.
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Substrate binding
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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.
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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)
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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
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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.)
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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.
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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
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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