Transcript Biochemistry 6/e - Personal Webspace for QMUL

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, relative to
catalysts in organic synthesis.
• They are exquisitely specific
Chymotrypsin
• Digestive enzyme secreted by the pancreas
• Serine protease
• Large hydrophobic amino acids
• Or specific for the peptide carbonyl
supplied by an aromatic residue (eg Tyr,
Met)
Specificity of chymotrypsin
Nucleophilic attack
Carbonyl bond
Hydrophobic amino acids
Common catalytic strategies
1. Covalent catalysis
• Reactive group (nucleophile)
2. General acid-base catalysis
•
proton donor/acceptor (not water)
3. Metal-ion catalysis
1. Nucleophile or electrophile eg Zn
4. Catalysis by approximation
1. 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
Half life for hydrolysis of typical peptide is 300600 years. Chymotrypsin accelerates the rate of
cleavage to 100 s-1 (>1012 enhancement).
Resonance
structure
The carbon-nitrogen bond is strengthened by its
double-bond character, and the carbonyl carbon
atom is less electrophilic and is less susceptible to
nucleophilic attack than are the carbonyl carbon
atoms in carboxylate esters.
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 Ophosphoserine
•
Sequencing established the serine to be Ser195
• Among 28 serines, Ser195 is highly reactive, why?
An unusually reactive serine in
chymotrypsin
Probing enzyme mechanism
Colourless
Carboxylic acid
Yellow product
Catalysed by chymotrypsin
Measure absorbance
Kinetics of chymotrypsin
catalysis
Covalent catalysis
Two stages
Stage 1- acylation
(p-nitrophenolate)
Deacylation through hydrolysis
Covalent
bond
Carboxylic acid
Location of the active site in
chymotrypsin
Hydrogen bonded
• His 57
• Asp 102
3 chains
• Catalytic Triad
The catalytic triad
Nucleophile
• Arrangement polarises serine hydroxyl group
• Histidine becomes a proton acceptor
• Stabilised by Aspartate
Peptide hydrolysis by
chymotrypsin
Step 1 – substrate binding
Nucleophilic
attack
2. Formation of the tetrahedral
intermediate
Ser 195
• -ve charge on oxygen stabilised
3. Tetrahedral intermediate
collapse
• Generates acyl-enzyme
– Transfer of His proton – amine component
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
WHY DOES CHYMOTRYPSIN
PREFER PEPTIDE BONDS
JUST PAST RESIDUES WITH
LARGE HYDROPHOBIC SIDE
CHAINS?
Specificity of chymotrypsin
Nucleophilic attack
Hydrophobic amino acids
Specificity pocket of
chymotrypsin (S1-pocket)
• Pocket Lined with hydrophobic residues
• Substrate side chain binding
– phenylalanine
S1-subsite
Specificity nomenclature for
protease – substrate interactions.
N-terminal
Scissile
bond
C-terminal
More complex specificity
P – potential sites of interaction with the enzyme (P’ – carboxyl side)
S – Corresponding binding site on the enzyme (specificity pocket)
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
Nucleophile
Eg Papain
Activation strategy
2.
Nucleophile
Asp
Asp
Eg Renin
Activation strategy
3.
Nucleophile
Water
Eg carboxypeptidase A
Activation strategy
Active site acts to either:-
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
Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition
Chapter 9:
Catalytic Strategies
Copyright © 2007 by W. H. Freeman and Company