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Transcript of Chymotrypsin Lecture Aims: to understand (1) the catalytic strategies used by enzymes and (2) the...
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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, relative to catalysts in organic synthesis.
• They are exquisitely specific
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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)
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Specificity of chymotrypsinNucleophilic attack
Hydrophobic amino acids
Carbonyl bond
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Common catalytic strategies1. Covalent catalysis• Reactive group (nucleophile)
2. General acid-base catalysis• proton donor/acceptor (not water)
3. Metal-ion catalysis1. Nucleophile or electrophile eg Zn
4. Catalysis by approximation1. 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, and the carbonyl carbon atom is less electrophilic and is less susceptible to nucleophilic attack than are the carbonyl carbon atoms in carboxylate esters.
Half life for hydrolysis of typical peptide is 300-600 years. Chymotrypsin accelerates the rate of cleavage to 100 s-1 (>1012 enhancement).
Resonancestructure
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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 waterHydroxyl 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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NHgroups
Stabilisation of intermediates
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WHY DOES CHYMOTRYPSIN PREFER PEPTIDE BONDS JUST PAST RESIDUES WITH LARGE HYDROPHOBIC SIDE CHAINS?
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Specificity of chymotrypsinNucleophilic 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 pocketsconfer 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 sites1.
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Proteases and their active sites2.
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Proteases and their active sites3.
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Activation strategy1.
His
Cys
Eg Papain
Nucleophile
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Activation strategy2.
Asp Asp
Eg Renin
Nucleophile
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Activation strategy3.
Eg carboxypeptidase A
Nucleophile
Water
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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.
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Protease inhibitors are important drugs
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HIV proteaseDimeric aspartyl protease
• Cleaves viral proteins– activation
Aspartateresidues
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HIV protease inhibitor
symmetry
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HIV protease-indovir complex
Asp
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BiochemistrySixth Edition
Chapter 9:Catalytic Strategies
Copyright © 2007 by W. H. Freeman and Company
Berg • Tymoczko • Stryer