chymotrypsin lecture aims: to understand (1) the catalytic strategies used by enzymes and (2) the...

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 chymotrypsinNucleophilic attack

Hydrophobic amino acids

Carbonyl bond

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

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, 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

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 waterHydroxyl ion attacks carbonyl

7. Formation of carboxylic acid product

8. Release of carboxylic acid

NHgroups

Stabilisation of intermediates

WHY DOES CHYMOTRYPSIN PREFER PEPTIDE BONDS JUST PAST RESIDUES WITH LARGE HYDROPHOBIC SIDE CHAINS?

Specificity of chymotrypsinNucleophilic 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 pocketsconfer 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 sites1.

Proteases and their active sites2.

Proteases and their active sites3.

Activation strategy1.

His

Cys

Eg Papain

Nucleophile

Activation strategy2.

Asp Asp

Eg Renin

Nucleophile

Activation strategy3.

Eg carboxypeptidase A

Nucleophile

Water

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 proteaseDimeric aspartyl protease

• Cleaves viral proteins– activation

Aspartateresidues

HIV protease inhibitor

symmetry

HIV protease-indovir complex

Asp

BiochemistrySixth Edition

Chapter 9:Catalytic Strategies

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer