lecture 13: mechanism of chymotrypsin chemical mechanism of chymotrypsin

Lecture 13:Mechanism of Chymotrypsin

Chemical Mechanism

of Chymotrypsin

Chymotrypsin

Chymotrypsin is a digestive protease involved in breakdown ofproteins and peptides so that their amino acids can be used.

It is synthesized in the pancreas of mammals and released intothe digestive tract.

When first synthesized it is as a single polypeptide chainin an inactive form, chymotrypsinogen, which must be activatedbefore the enzyme can fulfill its role.

Activation of chymotrypsin is achieved by “clips” in its polypeptidechain, so active chymotrypsin consists of three distinct chains.These remain bound together in a single domain, covalently heldtogether by disulfide bonds.

The activity of chymotrypsin is regulated by controlling when the“clips” are made.

Acid + AmineAmide + H2OOverall G of hydrolysis is negative (favorable).

G’

Gcat

Guncat

Reactants

Products

Uncatalysedpathway

Enzyme-catalysedpathway

Net Reaction:

Overview:

Substrate (S) binds.

Phase 1:Enzyme creates nucleophile from serine side-chain.Nucleophile attacks substrate.Covalent intermediate is formed with second product ( PN )

bonded to serine, and first product ( PC ) is released.

Phase 2:Enzyme creates a nucleophile from a water molecule.Nucleophile attacks covalent intermediate, breaking covalent bond to serine.

Second product ( PN ) is released.

Substrate Binding

E ES

ChemicalRearrangement

Product #1Released

EPN

ChemicalRearrangement

E-PN PC E-PN

(Bond to be cleaved.)PN PC

Product #2Released

S

PC PNS

G’

Gcat

Reactants

Products

ESE-PNPC

EPN

E+S ES E-PNPC E-PN

+ PC

EPN

+ PC

E+PN

+PC

Clues about mechanism:

Burst phase indicates a covalent intermediate is formed.

Kinetics experiments are used to figure out howmany steps there are in a reaction mechanismand how long each step takes.

Chemical labeling with DIPF finds one particular serine residue (out of 28)that is extremely reactive.

Chemical labeling experiments are used to figureout which residues are responsible for importantsteps in a reaction mechanism.

Crystal structure analysis reveals a catalytic triad, a group of 3 side-chainswhich are responsible for the peculiar reactivity of this serine.

Determination of the crystal structure of an enzymeprovides a detailed description of the three-dimensionalarrangement of the molecule and in particular of the activesite.

Chymotrypsin Kinetics

Very early in reaction,p-nitrophenolate is released giving rise tothe burst phase.

Subsequent reactantsmust wait for an activesite to become availablethrough release of anintermediate, givingrise to thesteady-state phase.

(remains covalentlybound)

Km = 20 Mkcat = 77 s-1

(released immediately)

Labelling of Serine 195 Inactivates Chymotrypsin

DIPF, an irreversible inhibitor, is a group-specific reagent for serineresidues. It forms a covalent adduct on Serine 195, which rendersthe enzyme inactive. Only Ser 195, out of 28 serines in chymotrypsin,is labelled, suggesting it is both especially reactive and that thisreactivity is necessary for catalysis.

Structure of Chymotrypsin

Globular single-domain protein.

Originally synthesized as a 245residue protein, chymotrypsinogen.

Dipeptides 14-15 and 147-148 areclipped out, tranforming the proteininto active chymotrypsin.

Therefore it has 3 chains (red, blue,green) but these are covalently linkedby disulfide bridges.

The reactive serine 195 is locatedin a cleft on the molecule, theactive site. Ser 195 is adjacent toHis 57 and Asp 102 which areresponsible for its reactivity.

Substrate (S) binds.

Enzyme creates nucleophile from serine side-chain.Nucleophile attacks substrate.Acyl-enzyme is formed with second product ( PN ) bonded to serine, and first product ( PC ) is released.

Enzyme creates a nucleophile from a water molecule.Nucleophile attacks acyl linkage, breaking covalent bond to serine.

Second product ( PN ) is released.

Phase 1:

Phase 2:

Substrate binding

Creation of NucleophileA nucleophile is a highly reactive, electron-rich group.

In chymotrypsin, serine 195 is converted into an alkoxide ion, a powerful nucleophile, through removal of its hydroxyl proton.

This difficult task is accomplished by the charge relay system between Asp 102, His 57, and Ser 195, which comprise the catalytic triad. His 57 can alternately accept or donate protons, whilestabilized by Asp 102.

(This is a good example of a general base in catalysis.)The charges are stabilized by electrostatic effects.

Nucleophilic Attack

The carbonyl carbon on the substrate has 3 bonds and so is a trigonal atom.

The alkoxide ion attacks the carbonyl carbon, forming a tetrahedral intermediate with 4 bond to that carbon.

The former carbonyl oxygen is converted into a negatively charged group,the oxyanion, which is stabilized by by an arrangement of partial positivecharges nearby in the oxyanion hole. (an electrostatic effect)

Formation of Acyl-enzyme

The tetrahedral intermediate breaks down when the histidine donates aproton and creates a new amino group on the terminus of the firstproduct ( PC ), which is released. (His 57 is acting as general acid.)

The remainder of the substrate remains attached to the enzyme throughan ester linkage to Serine 195. (Covalent catalysis.)

Acyl group

Creation and Use of New Nucleophile

Another nucleophile is created by the enzyme, using His 57 to withdrawa proton from a water molecule to form a hydroxide ion. (another exampleof general base catalysis)

This nucleophile attacks the acyl carbon forming a second tetrahedralintermediate, which is again stabilized by the oxyanion hole. (Anotherexample of the electrostatic effect.)

De-acylation Step

The tetrahedral intermediate breaks down when His 57 donates a protonto serine 195, displacing the acyl group and regenerating the serinehydroxyl group. The second product ( PN ) is released, concluding

the reaction.

Same Chemistry, Different Enzymes

Other unrelated classes of proteases, whose sequences and structuresare unrelated to those of chymotrypsin, nevertheless have the same spatial arrangement of the His-Asp-Ser catalytic triad.

The same catalytic method appears to have arisen independently at least three times in nature- an example of convergent evolution.

Subtilisin

Carboxypeptidase A

Summary:

Chymotrypsin is a protease and its activity is regulated by controlledcleavage of its backbone.

Its chemical mechanism proceeds in two stages:

1. Nucleophilic attack on substrate by Ser 195 to form acyl-enzymecomplex

followed by

2. Deacylation though nucleophilic attack by water on the acyl intermediate.

Key Concepts:

Meaning of burst phase and labelling of Serine 195Catalytic triad: Roles of His 57, Asp 102, and Ser 195 in mechanismOccurrences of acid-base catalysis and covalent catalysis in mechanism