Chymotrypsin Lecture
Aims: to understand (1) the catalytic
strategies used by enzymes and (2)
the mechanism of chymotrypsin
What’s so great about enzymes?
• They accomplish large rate accelerations
(1010-1023 fold) in an aqueous environment
using amino acid side chains and cofactors
with limited intrinsic reactivity
• They are exquisitely specific
Chymotrypsin
• Digestive enzyme secreted by the pancreas
• Serine protease
• Large hydrophobic amino acids
• Specific for the peptide carbonyl supplied
by an aromatic residue (eg Tyr, Met)
Specificity of chymotrypsin Nucleophilic attack
Hydrophobic amino acids
Carbonyl bond
Common catalytic strategies 1. Covalent catalysis
• Reactive group (nucleophile)
• Hydroxide ion
2. General acid-base catalysis
• proton donor/acceptor (not water)
3. Metal-ion catalysis
• Nucleophile or electrophile eg Zn
• Form bridge between enzyme and substrate
4. Catalysis by approximation
• Two substrates along a single binding surface
or, combination of these strategies eg an example of use of 1 & 2 is chymotrypsin
Proteases Catalyse a
Fundamentally Difficult Reaction
They cleave proteins by hydrolysis – the
addition of water to a peptide bond
• The carbon-nitrogen bond is strengthened by its double-bond character – carbonyl carbon atom is less electrophilic
– less susceptible to nucleophilic attack
– Enzyme must facilitate nucleophilic attack on normally unreactive carbonyl group
Half life for hydrolysis of typical peptide is 300-
600 years. Chymotrypsin accelerates the rate of
cleavage to 100 s-1 (>1012 enhancement).
Resonance
structure
Identification of the
reactive serine • Around 1949 the nerve gas di-isopropyl-fluorophosphate
was shown to inactivate chymotrypsin
• 32P-labelled DIPF covalently attached to the enzyme
• When labelled enzyme was acid hydrolysed the
phosphorus stuck tightly; the radioactive fragment was O-
phosphoserine
• Sequencing established the serine to be Ser195
• Among 28 serines, Ser195 is highly reactive, why?
An unusually reactive serine in
chymotrypsin
Probing enzyme mechanism
Catalysed by chymotrypsin Measure absorbance
Colourless
Yellow product
Carboxylic acid
Kinetics of chymotrypsin
catalysis
Covalent catalysis
Two stages
Stage 1- acylation
(p-nitrophenolate)
Deacylation through hydrolysis
Carboxylic acid
Covalent
bond
Location of the active site in
chymotrypsin
• His 57
• Asp 102
• Catalytic Triad
3 chains
Hydrogen bonded
The catalytic triad
• Arrangement polarises serine hydroxyl group
• Histidine becomes a proton acceptor
• Stabilised by Aspartate
Nucleophile
Peptide hydrolysis by
chymotrypsin
Step 1 – substrate binding
Nucleophilic
attack
Ser 195
2. Formation of the tetrahedral
intermediate
• -ve charge on oxygen stabilised
3. Tetrahedral intermediate
collapse
• Generates acyl-enzyme
– Transfer of His proton – amine component
formed
4.Release of amine component
(acylation of enzyme)
5. Hydrolysis
(deacylation)
6. Formation of tetrahedral
intermediate
Histidine draws proton from water
Hydroxyl ion attacks carbonyl
7. Formation of carboxylic acid
product
8. Release of carboxylic acid
NH
groups
Stabilisation of intermediates
(O2)
WHY DOES CHYMOTRYPSIN
PREFER PEPTIDE BONDS
JUST PAST RESIDUES WITH
LARGE HYDROPHOBIC SIDE
CHAINS?
Specificity of chymotrypsin Nucleophilic attack
Hydrophobic amino acids
S1-subsite
Specificity pocket of
chymotrypsin (S1-pocket)
• Pocket Lined with hydrophobic residues
• Substrate side chain binding
– phenylalanine
Specificity nomenclature for
protease – substrate interactions.
P – potential sites of interaction with the enzyme (P’ – carboxyl side)
S – Corresponding binding site on the enzyme (specificity pocket)
More complex specificity
Scissile
bond N-terminal C-terminal
S1 pockets
confer substrate specificity
Arg,lys
(+ve charge)
Ala, ser
(small side chain)
Subtilisin cf Chymotrypsin
Catalytic triad
Site directed mutagenesis
KM unchanged
Not all proteases utilise serine to
generate nucleophile attack
Proteases and their active sites
1.
Proteases and their active sites
2.
Proteases and their active sites
3.
Activation strategy
1.
His
Cys
Eg Papain
Nucleophile
Activation strategy
2.
Asp Asp
Eg Renin
Nucleophile
Activation strategy
3.
Eg carboxypeptidase A
Nucleophile
Activation strategy
Active site acts to :-
a) Activate a water molecule or other
nucleophile (cys, ser)
b) Polarise the peptide carbonyl
c) Stabilise a tetrahedral intermediate.
Protease inhibitors are important
drugs
HIV protease
Dimeric aspartyl protease
• Cleaves viral proteins
– activation
Aspartate
residues
HIV protease inhibitor
symmetry
HIV protease-indovir complex
Asp
Biochemistry Sixth Edition
Chapter 9:
Catalytic Strategies
Copyright © 2007 by W. H. Freeman and Company
Berg • Tymoczko • Stryer
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