chymotrypsin lecture - queen mary university of...
TRANSCRIPT
![Page 1: Chymotrypsin Lecture - Queen Mary University of Londonwebspace.qmul.ac.uk/rwjanes/basic_12_16_web.pdf · Chymotrypsin accelerates the rate of cleavage to 100 s-1 12(>10 enhancement)](https://reader033.vdocuments.mx/reader033/viewer/2022042804/5f52157f2dd2e156ed732db8/html5/thumbnails/1.jpg)
Chymotrypsin Lecture
Aims: to understand (1) the catalytic
strategies used by enzymes and (2)
the mechanism of chymotrypsin
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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
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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)
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Specificity of chymotrypsin Nucleophilic attack
Hydrophobic amino acids
Carbonyl bond
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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
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Proteases Catalyse a
Fundamentally Difficult Reaction
They cleave proteins by hydrolysis – the
addition of water to a peptide bond
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• 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
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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?
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An unusually reactive serine in
chymotrypsin
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Probing enzyme mechanism
Catalysed by chymotrypsin Measure absorbance
Colourless
Yellow product
Carboxylic acid
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Kinetics of chymotrypsin
catalysis
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Covalent catalysis
Two stages
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Stage 1- acylation
(p-nitrophenolate)
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Deacylation through hydrolysis
Carboxylic acid
Covalent
bond
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Location of the active site in
chymotrypsin
• His 57
• Asp 102
• Catalytic Triad
3 chains
Hydrogen bonded
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The catalytic triad
• Arrangement polarises serine hydroxyl group
• Histidine becomes a proton acceptor
• Stabilised by Aspartate
Nucleophile
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Peptide hydrolysis by
chymotrypsin
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Step 1 – substrate binding
Nucleophilic
attack
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Ser 195
2. Formation of the tetrahedral
intermediate
• -ve charge on oxygen stabilised
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3. Tetrahedral intermediate
collapse
• Generates acyl-enzyme
– Transfer of His proton – amine component
formed
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4.Release of amine component
(acylation of enzyme)
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5. Hydrolysis
(deacylation)
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6. Formation of tetrahedral
intermediate
Histidine draws proton from water
Hydroxyl ion attacks carbonyl
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7. Formation of carboxylic acid
product
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8. Release of carboxylic acid
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NH
groups
Stabilisation of intermediates
(O2)
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WHY DOES CHYMOTRYPSIN
PREFER PEPTIDE BONDS
JUST PAST RESIDUES WITH
LARGE HYDROPHOBIC SIDE
CHAINS?
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Specificity of chymotrypsin Nucleophilic attack
Hydrophobic amino acids
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S1-subsite
Specificity pocket of
chymotrypsin (S1-pocket)
• Pocket Lined with hydrophobic residues
• Substrate side chain binding
– phenylalanine
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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
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S1 pockets
confer substrate specificity
Arg,lys
(+ve charge)
Ala, ser
(small side chain)
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Subtilisin cf Chymotrypsin
Catalytic triad
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Site directed mutagenesis
KM unchanged
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Not all proteases utilise serine to
generate nucleophile attack
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Proteases and their active sites
1.
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Proteases and their active sites
2.
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Proteases and their active sites
3.
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Activation strategy
1.
His
Cys
Eg Papain
Nucleophile
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Activation strategy
2.
Asp Asp
Eg Renin
Nucleophile
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Activation strategy
3.
Eg carboxypeptidase A
Nucleophile
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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.
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Protease inhibitors are important
drugs
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HIV protease
Dimeric aspartyl protease
• Cleaves viral proteins
– activation
Aspartate
residues
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HIV protease inhibitor
symmetry
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HIV protease-indovir complex
Asp
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Biochemistry Sixth Edition
Chapter 9:
Catalytic Strategies
Copyright © 2007 by W. H. Freeman and Company
Berg • Tymoczko • Stryer