Chymotrypsin is an enzyme secreted in its inactive form, chymotrypsinogen, by the acinar cells of the pancreas. It is activated by another pancreatic enzyme, trypsin. Chymotrypsin is a proteolytic enzyme that is principally derived from ox pancreas. Chymotrypsin is synthesized by protein biosynthesis as a precursor called chymotrypsinogen that is enzymatically inactive. On cleavage by trypsin into two parts that are still connected via an S-S bond, cleaved chymotrypsinogen molecules can activate each other by removing two small peptides in a trans-proteolysis. The resulting molecule is chymotrypsin, three polypeptides interconnected via S-S bonds.

Chymotrypsin cleaves peptides and polypeptides to shorter peptide chains, tri- and dipeptides. Chymotrypsin preferentially catalyzes the hydrolysis of peptide bonds involving L-isomers of tyrosine, phenylalanine and tryptophan. It also readily acts upon amides and esters of susceptible amino acids. Chymotrypsin catalyzes the hydrolysis of bonds of leucyl, methionyl, asparaginyl and glutamyl residues. Chymotrypsin is a serine proteinase, referring to the fact that serine and histidine residues at the active site are involved in catalysis. Trypsin, also a serine proteinase, and chymotrypsin have similar tertiary structures although very different substrate specificities. Trypsin hydrolyzes peptides at Lys/Arg residues while chymotrypsin recognizes large hydrophobic residues. Chymotrypsin is found in pancreatic preparations, such as pancreatin and pancrelipase. It is used in ophthalmology for the dissection of the zonule of the lens. It is also used as a digestant and it has putative anti-inflammatory activity.

Although an enzyme called pepsin begins to digest protein molecules while they are in our stomach, most of the digestion of protein takes place after the food leaves the stomach and moves into the small intestines. Here enzymes like trypsin, chymotrypsin, elastase, carboxypeptidase, and aminopeptidase convert protein molecules into amino acids, dipeptides, and tripeptides. The dipeptides and tripeptides are converted to amino acids by other enzymes. Once the amino acids are free, they can move into the blood stream and circulate throughout our body.

This enzyme catalyses the hydrolysis of peptide bonds of proteins in the small intestine. It is selective for peptide bonds with aromatic or large hydrophobic side chains (Tyr, Trp, Phe, Met) on the carboxyl side of this bond. Chymotrypsin also catalyses the hydrolysis of ester bonds. X-ray studies have revealed a `charge relay system' of Asp-102, His-57 and Ser-195. This grouping has been found in a whole group of enzymes called the `serine proteases'. Neutron diffraction studies on trypsin show that His-57 acts as a base in the catalytic process. The hydrolysis of peptide bonds occurs by general base-catalysed nucleophilic attack on the carbonyl carbon of the substrate by the hydroxyl oxygen of Ser-195. At the same time the hydroxyl proton of serine is transferred to the imidazole of His-57, the chemical base in the hydrolysis reaction.