CHYMOTRYPSIN: CHARACTERISTICS, STRUCTURE, FUNCTIONS, MECHANISM OF ACTION - BIOLOGY - 2025

The chymotrypsin is the second most abundant digestive protein secreted by the pancreas into the small intestine. It is an enzyme belonging to the family of serine proteases and specializes in the hydrolysis of peptide bonds between amino acids such as tyrosine, phenylalanine, tryptophan, methionine and leucine present in large proteins.

The name “chymotrypsin” actually brings together a group of enzymes that are produced by the pancreas and that actively participate in the intestinal digestion of proteins in animals. The word derives from the renin-like action that this enzyme has on gastric contents or the “chyme”.

Chymotrypsin structure (Source: User: Mattyjenjen via Wikimedia Commons)

Although it is not known exactly how wide their distribution is in the animal kingdom, it is considered that these enzymes are present at least in all chordates and there are reports of their presence in "more primitive phyla" such as arthropods. and that of the coelenterates.

In those animals that have a pancreas, this organ is the main site of chymotrypsin production, as well as other proteases, enzyme inhibitors, and precursors or zymogens.

Chymotrypsins are the most studied and best characterized enzymes, not only in relation to their biosynthesis, but also to their activation from zymogen, their enzymatic properties, their inhibition, their kinetic and catalytic characteristics, and their general structure.

Characteristics and structure

Chymotrypsins are endopeptidases, that is, they are proteases that hydrolyze peptide bonds of amino acids in "internal" positions of other proteins; although it has also been shown that they can hydrolyze esters, amides and arylamides, although with less selectivity.

They have an average molecular weight of about 25 kDa (245 amino acids) and are produced from precursors known as chymotrypsinogens.

From the pancreas of bovine animals, 2 types of chymotrypsinogens have been purified, A and B. In the porcine model, a third chymotrypsinogen was described, chymotrypsinogen C. Each of these three zymogens is responsible for the production of chymotrypsins A, B and C, respectively.

Chymotrypsin A is made up of three polypeptide chains that are covalently linked to each other through bridges or disulfide bonds between cysteine ​​residues. However, it is important to mention that many authors consider it a monomeric enzyme (composed of a single subunit).

These chains compose a structure that has an ellipsoid shape, in which the groups that have electromagnetic charges are located towards the surface (with the exception of the amino acids that participate in catalytic functions).

Chymotrypsins are generally highly active at acidic pHs, although those that have been described and purified from insects and other non-vertebrate animals are stable at pH 8-11 and extremely unstable at lower pH.

Chymotrypsin functions

When the exocrine pancreas is stimulated, either by hormones or by electrical impulses, this organ releases secretory granules rich in chymotrypsinogen, which, once it reaches the small intestine, is cut by another protease between residues 15 and 16 and then is " self-processed ”to yield a fully active protein.

Perhaps the main function of this enzyme is to act in concert with the other proteases excreted into the gastrointestinal system for the digestion or degradation of proteins consumed with food.

The products of said proteolysis subsequently serve as a source of carbon and energy through the catabolism of amino acids or they can be "recycled" directly for the formation of new cellular proteins that will exert multiple and varied functions at the physiological level.

Mechanism of action

Chymotrypsins exert their actions only after being activated, as these are produced as "precursor" forms (zymogens) called chymotrypsinogens.

Chymotrypsin reaction mechanism (Source: Hbf878 via Wikimedia Commons)

Training

Chymotrypsin zymogens are synthesized by the acinar cells of the pancreas, after which they migrate from the endoplasmic reticulum to the Golgi complex, where they are packaged within membranous complexes or secretory granules.

These granules accumulate at the ends of the acini and are released in response to hormonal stimuli or nerve impulses.

Activation

Depending on the activation conditions, several types of chymotrypsins can be found, however, all of them involve the proteolytic “cleavage” of a peptide bond in the zymogen, chymotrypsinogen, a process catalyzed by the enzyme trypsin.

The activation reaction initially consists of the cleavage of the peptide bond between amino acids 15 and 16 of chymotrypsinogen, with which π-chymotrypsin is formed, capable of “self-processing” and completing the activation by autocatalysis.

The action of the latter enzyme promotes the formation of subsequent peptides linked by disulfide bonds and these are known as chain A (from the N-terminal region and residues 1-14), chain B (residues 16 to 146) and the C chain (C-terminal region, beginning with residue 149).

The portions corresponding to residues 14-15 and 147-148 (two dipeptides) do not have catalytic functions and are detached from the main structure.

Catalytic activity

Chymotrypsin is responsible for hydrolyzing peptide bonds, predominantly attacking the carboxylic portion of amino acids that have aromatic side groups, that is, amino acids such as tyrosine, tryptophan, and phenylalanine.

A serine (Ser 195) within the active site (Gly-Asp-Ser-Gly-Glu-Ala-Val) of this type of enzyme is perhaps the most essential residue for its functioning. The reaction mechanism is as follows:

- Chymotrypsin is initially in a “substrate-free” form, where the catalytic “triad” consists of the side carboxyl group of an aspartate residue (102), the imidazole ring of a histidine residue (57) and the side hydroxyl group of a serine (195).

- The substrate meets the enzyme and binds to it to form a typical reversible enzyme-substrate complex (according to the mycaelian model), where the catalytic "triad" facilitates nucleophilic attack by activating the hydroxyl group of the serine residue.

- The key point of the reaction mechanism consists in the formation of a partial bond, which results in the polarization of the hydroxyl group, which is sufficient to accelerate the reaction.

- After nucleophilic attack, the carboxyl group becomes a tetrahedral oxyanion intermediate, which is stabilized by two hydrogen bonds formed by the N and H groups of the residue of Gly 193 and Ser 195.

- The oxyanion spontaneously "rearranges" and forms an enzyme intermediate to which an acyl group (acylated enzyme) has been added.

- The reaction continues with the entry of a water molecule to the active site, a molecule that promotes a new nucleophilic attack that results in the formation of a second tetrahedral intermediate that is also stabilized by hydrogen bonds.

- The reaction ends when this second intermediate rearranges again and forms the enzyme-substrate micaelian complex again, where the active site of the enzyme is occupied by the product that contains the carboxyl group.

References

1. Appel, W. (1986). Chymotrypsin: molecular and catalytic properties. Clinical biochemistry, 19 (6), 317-322.
2. Bender, ML, Killheffer, JV, & Cohen, S. (1973). Chymotrypsin. CRC critical reviews in biochemistry, 1 (2), 149-199.
3. Blow, DM (1971). 6 The Structure of Chymotrypsin. In The enzymes (Vol. 3, pp. 185-212). Academic Press.
4. Blow, DM (1976). Structure and mechanism of chymotrypsin. Accounts of chemical research, 9 (4), 145-152.
5. Nelson, DL, Lehninger, AL, & Cox, MM (2008). Lehninger principles of biochemistry. Macmillan.
6. Polgár, L. (2013). Catalytic mechanisms of serine and threonine peptidases. In Handbook of Proteolytic Enzymes (pp. 2524-2534). Elsevier Ltd.
7. Westheimer, FH (1957). Hypothesis for the mechanism of action of chymotrypsin. Proceedings of the National Academy of Sciences of the United States of America, 43 (11), 969.