Download Chymotrypsin

Hypothetical substrate docking in enzyme’s
active site. Substrate is geometrically and
electronically compatible with active site.
Enzymes are also stereospecific.
ES complex formation
How is
‡
G
lowered?
Many enzymes stabilize the transition state in addition
to the mechanisms described below through
relatively tight binding.
Mechanisms of catalysis
1. Acid-Base catalysis
2. Covalent catalysis
3. Metal ion catalysis
4. Proximity and orientation effects
5. Electrostatic catalysis
1. Acid-Base catalysis (H+ transfer between E and S)
Acid catalysis
Base catalysis
2. Covalent catalysis (covalent bond between E and S)
Amine in enzyme reacts with carbonyl group to form Schiff base
Elimination of CO2 then does not lead to an unstable transition state
Schiff base breaks down to amine catalyst and acetone
3. Metal ion catalysis (metal ions can mediate redox
reactions or electrostatically promote reactions).
Proximity/Orientation
No catalyst: slow
b/c few encounters
Catalyst: faster
5. Electrostatic catalysis
Most substrate molecules have a hydration shell that must be removed to allow reaction.
Enzyme active sites are often non-aqueous (not necessarily non-polar) and they require
that water is removed from the substrate before binding can occur.
The non-aqueous active site allows stronger electrostatic interactions.
Chymotrypsin
Digestive enzyme synthesized in
pancreas and secreted to small
intestine.
Has broader specificity than most
enzymes.
Hydrolyzes the peptide bond
following large nonpolar residues
like Phenylalanine, Tryptophan or Leu-Ala-Tyr-Ile-Asp
Chymotrypsin. 241 amino acid protein with 2 domains and an active
Tyrosine.
site with 3 residues important for catalysis shown in red (His 57, Asp
102 and Ser 195).
Chymotrypsin is part of a class of enzymes known as
Serine Proteases.
Uses acid-base and covalent catalysis, and it stabilizes
the transition state.
His 57
Asp 102
Ser 195
Let’s look at the detailed reaction mechanism
for chymotrypsin, a protease:
It hydrolyzes peptide bonds immediately
C-terminal to aromatic amino acids
With a rate enhancement of > 109
And illustrates several principles
Transition state stabilization
Acid-base catalysis
Covalent catalysis
Leu-Ala-Tyr-Ile-Asp
A two-phase reaction
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Appreciate the contribution of
protein folding
PDB: 7GCH
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Mechanism in the
Substrate Pocket
• Phase I - acylation of
Ser195
• Phase 2 – deacylation of
Ser195
• Follow the roles of the
“catalytic triad” through
the reaction: Ser195, His57
and Asp102
• What is the role of
Gly193?
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In the active site of chymotrypsin, know
the chemical roles of:
•
•
•
•
Histidine 57
Aspartate 102
Serine 195
Glycine 193
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Chymotrypsin Mechanism -1
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Chymotrypsin Mechanism -2
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Chymotrypsin Mechanism - 3
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Chymotrypsin Mechanism - 4
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Chymotrypsin Mechanism - 5
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Chymotrypsin Mechanism - 6
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Chymotrypsin Mechanism - 7
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Chymotrypsin Catalytic Mechanism A1
Catalytic Triad
His57
Asp102
Ser195
H
H
[HOOC]
C
N
C
N
H
O
C
C
O
Check substrate specificity
C
N
[NH2]
C
Chymotrypsin Catalytic Mechanism A2
His57
Asp102
Ser195
H
H
O
First Transition State
Chymotrypsin Catalytic Mechanism A3
H
H
O
Acyl-Enzyme Intermediate
Chymotrypsin Catalytic Mechanism D1
H
O
Acyl-Enzyme Water Intermediate
Chymotrypsin Catalytic Mechanism D2
H
O
H
O
Second Transition State
Chymotrypsin Catalytic Mechanism D3
H
O
H
O
Deacylation