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