Chymotrypsin: Structure, Activation, and Function

• Chymotrypsin is a protease, an enzyme that cuts polypeptides into smaller fragments.
• It has a molecular weight of 27 kD and contains 245 amino acids.
• The 3D structure was determined by crystallography.
• The structure is predominantly ẞ-sheets with a few a-helices on the periphery.

Enzyme and its physiology

• The protein is produced as a precursor called chymotrypsinogen that is enzymatically inactive.
• Chymotrypsinogen is cleaved by trypsin but held together by disulfide bonds.
• Final activation is achieved by cutting off a short peptide by partly activated chymotrypsin molecules.
• Elaborate activation mechanism is necessary to protect the organism from self-digestion.
• Chymotrypsin is a member of the family of serine proteases like trypsin, thrombin and elastase.
• It is produced in the pancreas and is used in the digestive system (small intestine) to help in breaking down food.
• Chymotrypsin has a preference to cleave peptide bonds near large hydrophobic amino acids.
• Hydrolysis of peptide bonds is thermodynamically favorable ('spontaneous') but very slow in the absence of catalysts.

Reaction mechanism of peptide bond cleavage

• Chymotrypsin catalyses the hydrolytic cleavage of a peptide bond.
• A peptide bond is chemically an amide and the reaction requires a molecule containing an amide and water.
• The reaction type is a nucleophilic substitution in which the R-NH group is replaced by an OH group turning the amide into an acid and an amine.
• The reaction starts by the formation of a covalent intermediate with the reactive centre, serine 195 (Ser195, S195).
• Normally alcohols have very high pKa values (16 for serine & threonine), dissociation is a very rare event, and the alkoxide anion is very unstable.
• In the active site the alkoxide O- group is stabilised in the "catalytic triad" of an adjacent histidine which acts as a base and in turn is stabilised by hydrogen bonds to a nearby aspartate.

Catalytic triad

• The catalytic triad consists of Asp 102, His 57 and Ser 195.

Oxyanion hole

• The oxyanion hole stabilises the negative charge by hydrogen bonds to backbone amide hydrogens.
• Hydrogens bound to nitrogen carry a partial positive charge which can help to balance the negative charge on the oxygen.
• A very tight turn surrounds the oxyanion.
• It requires a glycine.
• Backbone nitrogens are well positioned to provide hydrogen bond stabilisation to the oxyanion.

Substrate specificity

• Chymotrypsin hydrolyses specifically the peptide bond following large hydrophobic amino acids.
• Polypeptides can bind in "channels" along the surface.
• It is very flexible regarding substrate sequence as long as it has an extended conformation.
• Bulky sidechain in P1 position has to fit in S1 cavity.
• Wide range of charges are along channels.
• It is uncharged around the P1 site (white) to positive (blue) and negative (red).
• The range of different charged environments along different channels will stabilise a wide range of peptide sequences.

Experimental studies of chymotrypsin

• Model compounds are used to study enzyme kinetics.
• Their advantages are that they are visible, require no expensive optical instruments, can be synthesized in large quantities, have a long shelf life, and have a very simple structure simplifying interpretation of data.
• Two phases can be observed: a steady-state phase and a burst phase.
• The enzyme has a turnover number of 100 per second.
• Two phase kinetics gave the first evidence for two step mechanism.
• The burst phase occurs due to initial formation of acyl-enzyme intermediate is fast, hence swift emergence of yellow colour.
• The steady state phase is when the hydrolysis of acyl-enzyme intermediate occurs; this step is slower but important to recycle the enzyme to keep the reaction going.
• An acyl fragment is released from the enzyme via nucleophilic attack of water which is essentially split into OH- and H+ in the catalytic triad.
• By making the solution very acidic (pH 3) any OH- is immediately removed so that the acyl intermediate cannot be resolved, and the reaction stops.
• The acyl-enzyme complex can be isolated and studied e.g. by mass spectrometry to identify the amino acids to which the acyl group is attached.
• Enzyme kinetics combined with site directed mutagenesis and comparing kcat with wildtype and uncatalysed reaction gives good insight into the importance of different residues.
• This requires 3D structure.
• Reactive residues in the active site are identified by covalent labelling with substrate analogues.
• Normally amino acids are rather inert to undergo chemical reactions but a reaction is a sign of activation.
• The reaction has to result in stable product to facilitate analysis.
• Labelled protein can be further analysed by proteolytic cleavage using trypsin and mass spectrometry of the resulting protein fragments.
• A comparison with mass spectrometry data for the unmodified enzyme will allow the identification of the modified residue.
• The activated serine 195 is a powerful nucleophile that can perform a nucleophilic attack on the phosphorus atom of DIPF.
• In a nucleophilic substitution the fluoride leaves and the residual DIP remains covalently attached to the enzyme.
• The labelled residue can be identified using mass spectrometry.
• DIPF in contrast to TPCK is not a substrate analogue, but it simply collides with the activated serine by diffusion into the active site of the enzyme.
• DIPF will react with any enzyme that has an activated serine while TPCK will only react with enzymes that have an activated histidine and that have a substrate specificity for peptide bond cleavage near bulky, hydrophobic amino acids.
• To covalently modify an activated histidine in proteases with different substrate specificity the chemical have to be modified accordingly.

Summary

• A broad range of methods that are necessary to understand how the enzyme works.
• It's possible to precisely dissect the reaction mechanism in the enzyme and how it manages to act as a catalyst and achieve substrate specificity.
• Enzyme catalysed reactions proceed pretty much as in solution — the enzyme only gives a helping hand here and there.
• Example are acid and base catalysed reaction where the enzyme takes over the role of the water.
• The enzyme is much more effective than water because it can make these reagents available at the right time and the right place.