1. Chymotrypsin Lecture
Aims: to understand (1) the catalytic strategies used by enzymes and (2) the mechanism of chymotrypsin

2. What’s so great about enzymes?
They accomplish large rate accelerations ( 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

3. 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)
In this lecture we are going to concentrate on the mechanism of action of chymotrypsin. Cleaves peptide bonds on the carboxyl terminal side of hydrophobic amino acids such as tyrosine, or methionine

4. Specificity of chymotrypsin Nucleophilic attack
Carbonyl bond
Hydrophobic amino acids

5. Common catalytic strategies
Covalent catalysis
Reactive group (nucleophile)
General acid-base catalysis
proton donor/acceptor (not water)
Metal-ion catalysis
Nucleophile or electrophile eg Zn
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
Enzymes use one or more of the following strategies
Covalent catalysis – active site contains an active group (nucleophile) results in temporary covalent binding of substrate to active site – powerful nucleophil.
Approximation – as its name suggests it brings two reactants together along a single binding surface
Acid base catalysis - Molecule other than water plays a role of proton donor or acceptor.
Metal ion catalysis – acts as nucleophile
Catalysis by approximation -

6. Proteases Catalyse a Fundamentally Difficult Reaction
Protein turnover is important. Proteins must be degraded so that their amino acids can be recycled for the synthesis of new proteins. Proteases cleave by hydrolysis – addition of water across a peptide bond.
In absence of enzyme would take years. Yet in the body they are hydrolysed within milliseconds.
Double bond
They cleave proteins by hydrolysis – the addition of water to a peptide bond

7. Half life for hydrolysis of typical peptide is 300-600 years
Half life for hydrolysis of typical peptide is years. Chymotrypsin accelerates the rate of cleavage to 100 s-1 (>1012 enhancement).
Resonance
structure
Adopts a resonance structure – which as you may know prevents rotation about this bond and thus maintains the peptide bond in a planar conformation. Structure is strengthened by presence of double bond making carbonyl carbon less electrophilic and less susceptible to nucleophilic attack (addition of a pair of electrons).
To be able to perform this hydrolysis the enzyme must facilitate nucleophilic attackattack this carbonyl 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.

8. 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?
The answer is that the chymotrypsin contains an extraordinarily reactive serine in its structure which was identified by:
Chymotrypsin contains a reactive serine in the active site which acts as the nucleophile. What was the evidence for this? This all suggested that this unusually reactive serine plays a central role in the catalytic mechanism of chymotrypsin.

9. An unusually reactive serine in chymotrypsin
Here we can see DIPF binding to serine 195. Why this among 28 ser residues in the structure of chymotrypsin?

10. Probing enzyme mechanism
Colourless
To study the mechanism of action of this enzyme, kinetic studies wereperformed using a synthetic substrate that produces a coluored product and thus the reaction can be followede by detecting the appearance of the coloured productt.
N-acetyl-phenylalanine p-nitrophenyl ester is cleaved by chymotryspin which produces a yellow product p-nitrophenoloate.
Thus can follow the kinetics of this enzyme activity .
Carboxylic acid
Yellow product
Catalysed by chymotrypsin
Measure absorbance

11. Kinetics of chymotrypsin catalysis
Analysis showed that the reaction occurred in 2 phases. If measure absorbance with time using stopflow (measures vreactions in milliseconds)Two stages of activity are evident. A rapid burst of colored product followed by a slower formation as the reaction reaches steady state.

12. Covalent catalysis Two stages
Two stages are explained by hydrolysis taking place in two stages. Firstly acylation ie acyl group of substrate becomes covalently attached to the enzyme as paranitophenolate is released. the acyl-enzyme enzyme intermediate is the enzyme acyl complex. This is then hydrolysed to release the carboxylic acid component ofd the substrate and the enzyme is reformed
Two stages

13. Stage 1- acylation
(p-nitrophenolate)
Forms acyl enzyme intermediate

14. Deacylation through hydrolysis
Covalent
bond
Carboxylic acid

15. Location of the active site in chymotrypsin
His 57
Asp 102
Catalytic Triad
Hydrogen bonded
3 chains
3d structure solved in Found to Consists of three chains linked by disulphide bridges. Active site is a cleft on the surface of the enzyme. Serine is hydrogen bonded to his 57 which in turn is hydrogen bonded to asp 102. Referred to as the Catalytic Triad

16. The catalytic triad Arrangement polarises serine hydroxyl group
Histidine becomes a proton acceptor
Stabilised by Aspartate
Nucleophile
We can see this arrangement in more detail here. Triad converts serine into a potent nucleophile (electron pair donor) through withdrawal of proton from alcohol of serine.

17. Peptide hydrolysis by chymotrypsin
Mechanism of peptide hydrolysis takes place in eight steps.
Substrate binding
2. nucleophilic attack of serine on the peptide

18. Step 1 – substrate binding
Nucleophilic
attack
Substrate binding. Nucleophile attack

19. 2. Formation of the tetrahedral intermediate
-ve charge on oxygen stabilised
Ser 195
Oygen atom of serine 195 making nucleophile attack on the carbonyl carbon atom of the target peptide bond. 4 atoms bound to the carbonyl carbon arranged as a tetrahedron. Forms a negative charge on the oxygen atom which is stabiliosed by the interaction with the NH groups from the protein in a site termed the oxyanion hole. Stabilises the transition state.

20. 3. Tetrahedral intermediate collapse
Generates acyl-enzyme
Transfer of His proton – amine component formed
Negative charge on substarte stabilised by interaction with NH groups from a site in the protein termed the oxyanion hole Tyetrahedral intermediate collapses tom generate the acyl enzyme. Helped by transfer of the proton from the positively charged histidine residue to the amine formed by the cleavage of the peptide bond.

21. 4.Release of amine component (acylation of enzyme)
The amine component is now free to laeve the enzyme. Completing the first stage of the hydrolytic reaction – acylation of the enzyme

22. 5. Hydrolysis (deacylation)

23. 6. Formation of tetrahedral intermediate
Histidine withdraws a proton from the water molecule
Hydroxyl ion Another negatively charged oxygen is produced and stabilised by oxyanion hole
Histidine draws proton from water
Hydroxyl ion attacks carbonyl

24. 7. Formation of carboxylic acid product
This structure breaks down to form the carboxylic acid product.

25. 8. Release of carboxylic acid
Release of carboxylic acid and reformed enzymel. Peptide bond succesfully hydrolysed

26. Stabilisation of intermediatesNH
groups
The oxyanion hole is important in stabilising the intermediate tetrahedral through hydrogen bonds that link the negatively charged oxygen atom (shown in pink) to the NH groups shown in white.
Stabilisation of intermediates

27. Why does chymotrypsin prefer peptide bonds just past residues with large hydrophobic side chains?

28. Specificity of chymotrypsin Nucleophilic attack
Hydrophobic amino acids

29. Specificity pocket of chymotrypsin (S1-pocket)
Pocket Lined with hydrophobic residues
Substrate side chain binding
phenylalanine
Lined with relatively hydrophobic residues and relatively deep favouring bindingf of residues with long hydrophobic side chains. Binding of an appropriate side chain into this pocket positions the adjacent peptide bond into the active site for cleavage
S1-subsite

30. Specificity nomenclature for protease – substrate interactions.
Scissile bond
N-terminal
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)

31. S1 pockets confer substrate specificity
40% homology with chymotrypsin. Many enzymes have catalytic triads. These three enzymes have markedly different substrate specificity.
Trypsin cleaves at peptide after residues with long positively charged side chains Arg Lys
Elastase cleaves at the peptide bond after aminmo acids with small side chains alanine, serine
Look at S1 domains can see small structural differences. Trypsin contains an aspartate at the bottom of the pocket in place of serine. Stabilises a positive charge on arginine or lysine residue in substrate.
Elastase – two bulkier residues at top of pocket, close off mouth of pocket so that only small chains can enter.
Arg,lys
(+ve charge)
Ala, ser
(small side chain)

32. Subtilisin cf Chymotrypsin
Ymotrytpsin. How can we be sure the mechanism of the triad is correct. Can investigate subtilisin which has a similar structure to chSimilar to chymotrypsin in that it possesses both an oxyanion hole and cataklytic triad. Although one of the NH side chains comes from the side chain of an asparagine residue then the peptide backbone
Catalytic triad

33. Site directed mutagenesis
KM unchanged
Test role of individual amino acids to prove the importance of the catalytic triad by mutagenesis expets. Letter denotes amino acid and the number refers to the position of residue in the chain. Each of the triad are replaced with alanine and the catalytic activity is measured.
Km unchanged thus normal binding remains. Changing any or all effects the enzyme activity. His and ser appear to be the most important pair in nucleophilic attack. Mutated enzymes still 1000 times more effective than buffer alone (no enzyme)

34. Not all proteases utilise serine to generate nucleophile attack

35. Proteases and their active sites 1.
Not all proteases use serine residues. Three alternative approaches. They all generate a nucleophile that attacks the peptide

36. Proteases and their active sites 2.

37. Proteases and their active sites 3.

38. Activation strategy 1. His Cys Nucleophile Eg Papain
Histidine residue plays the role of nucleophile that attacks the peptide bond
Nucleophile
Eg Papain

39. Activation strategy 2. Nucleophile Asp Asp Eg Renin
Pair of aspartic acids work together to allow a water molecule to attack the peptide bond.
One activates the water by poising it for deprotonation. The other polarises the peptide carbonyl group so that it is more susceptible to attack.
Eg Renin

40. Activation strategy 3. Nucleophile Water Eg carboxypeptidase A
Bound metal ion (usually Zinc) activates a water molecule to act as nucleophile to attack the peptide carbonyl group
Eg carboxypeptidase A

41. Activation strategy Active site acts to either:-
Activate a water molecule or other nucleophile (cys, ser)
Polarise the peptide carbonyl
Stabilise a tetrahedral intermediate.

42. Protease inhibitors are important drugs

43. HIV protease Dimeric aspartyl protease
Cleaves viral proteins
activation
Flaps close down on the substrate after it has bound
Aspartate
residues

44. HIV protease inhibitor
symmetry
Scissile bond - covalent chemical bond that can be broken by an enzyme
The idovir adopts a conformation that approximates the two fold symmetry of the enzyme

45. HIV protease-indovir complex
Central alcohol interacts with both aspartate residues of the active site
Asp

46. Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition
Chapter 9:
Catalytic Strategies
Copyright © 2007 by W. H. Freeman and Company