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) 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

Specificity of chymotrypsin Nucleophilic attack Carbonyl bond Hydrophobic amino acids

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 -

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 10-1000 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

Half life for hydrolysis of typical peptide is 300-600 years 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 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.

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.

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?

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

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.

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

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

Deacylation through hydrolysis Covalent bond Carboxylic acid

Location of the active site in chymotrypsin His 57 Asp 102 Catalytic Triad Hydrogen bonded 3 chains 3d structure solved in 1967. 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

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.

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

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

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.

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.

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

5. Hydrolysis (deacylation)

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

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

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

Stabilisation of intermediates NH 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

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 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

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)

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)

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

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)

Not all proteases utilise serine to generate nucleophile attack

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

Proteases and their active sites 2.

Proteases and their active sites 3.

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

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

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

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

Protease inhibitors are important drugs

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

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

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

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