Serine Proteases: Chymotrypsin


Chymotrypsin has about 245 residues. It is a compact, globular protein composed of two domains, both of which have extensive antiparallel b sheets arranged like barrels. Chymotrypsin's compact structure makes it a stable enzyme.


In chymotrypsin, the active site serine is residue 195.

There are two other residues that lie near serine 195 that are also conserved among the serine proteases, namely, histidine 57 and aspartate 102. Together, these three residues make up the catalytic triad.

Review question 1

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Click on an atom of the amino acid that is the namesake for chymotrypsin's family of proteins.

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Active site

The enzyme's active site is located in a solvent-inaccessible pocket. This pocket also contains residues that are important for the specificity of the chymotrypsin enzyme. Chymotrypsin prefers to cleave next to bulky, hydrophobic amino acids. The binding pocket of chymotrypsin has a deep hole formed by hydrophobic side chains that accommodates the side chain next to the peptide bond that is to be cleaved.

Other serine proteases retain the Asp-His-Ser triad, but have different residues in this pocket conferring different specificities for binding and cleavage. For example, trypsin has a deeper specificity pocket that contains a negatively charged side chain at the bottom. Trypsin therefore favors the binding of positively charged residues, and generally cleaves a polypeptide after an arginine or lysine.

Subtilisin, found in the bacterium Bacillus subtilis, is a third example of a serine protease. It has an overall topology that is totally different from trypsin or chymotrypsin, but its active site Asp, His, and Ser residues are placed in almost identical positions relative to each other in the active site. This appears to be a case of convergent evolution.

The oxyanion hole

The binding site specificity pocket and the catalytic triad are important components of the chymotrypsin chemical machinery. However, another important feature is the oxyanion hole, located near the carbonyl group of the substrate's scissile bond. The name "oxyanion hole" describes a region in space where the backbone amide hydrogens of serine 195 and glycine 193 point into the active site cavity. These amino groups (area outlined in dots) are positioned in such a way that a tetrahedral enzyme–substrate intermediate is stabilized. The oxyanion hole increases the activity of the enzyme by a factor of 10,000.

Review question 2:

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Click on the atom of the inhibitor that occupies the oxyanion hole.

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Uncatalyzed hydrolysis

The reaction catalyzed by chymotrypsin is a simple hydrolysis of the peptide bond. The bond is broken by the addition of a water molecule to the peptide bond. An OH is added to the carbonyl group at the N-terminal end of the bond, and a proton (H+) is added to the amino group at the C-terminal end of the bond.

Chymotrypsin mechanism animation

Enzyme-catalyzed hydrolysis takes place in six-steps:

  1. Substrate binding
  2. Formation of tetrahedral intermediate
  3. Formation of the acyl-enzyme intermediate
  4. Departure of the amine product and arrival of H2O
  5. Deacylation
  6. Departure of the carboxylate product

Chymotrypsin mechanism structural exploration

We have seen the general mechanism of chymotrypsin in a two-dimensional sketch. This is a useful way to learn about the salient features of the mechanism, but let us take a look at the reaction as it proceeds inside the active site of chymotrypsin.

The imidazole nitrogen of histidine 57 abstracts the proton from serine 195. This leads to the electron transfers that result in the tetrahedral intermediate. The oxyanion hole stabilizes the tetrahedral intermediate.

The tetrahedral intermediate collapses and the acyl intermediate is formed. The first reaction product, an amine, departs.

After the amine leaves, a water molecule enters and repeats the process, breaking the acyl intermediate and forming a second tetrahedral intermediate.

The newly formed shortened peptide is ready to depart, concluding the catalytic cycle of chymotrypsin.

Chymotrypsinogen activation

In the intestine, newly synthesized chymotrypsinogen from the duodenum must first be proteolytically cleaved to form the active chymotrypsin shown here. This initial cleavage is performed by another serine protease, trypsin, followed by further self-activation by the resulting chymotrypsin. This activation by proteolysis lessens the strain in the active site, allowing the formation of an active conformation.

Review question 3

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Choose the residues of the catalytic triad.

Review question 4

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Identify the following parts of the chymotrypsin active site.

Review question 5

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Select the appropriate stage(s) of peptide hydrolysis that is (are) stabilized by the oxyanion hole.

You have completed this exercise.