Trypsin

Trypsin (EC 3.4.21.4) is a pancreatic serine protease from the S1 family and PA clan superfamily. Trypsin is a proteolytic enzyme that located in the digestive system, which is important for the digestion of proteins. In humans, trypsin is produced initially in its inactive form, trypsinogen, within the pancreas. Then, trypsinogen enters the small intestine and converted to active trypsin. The main function of trypsin is cleaving peptide chains at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. The two main forms of trypsin are α-trypsin and β-trypsin.

Structure

Bovine trypsinogen consists of a single polypeptide chain of 229 amino acids and is cross linked by six disulfide bridges. Trypsin consists of a single polypeptide chain of 223 amino acids. The active site of trypsin include His46 and Ser183. The native form of trypsin is referred to as β-trypsin, which can be autolyzed to form α-trypsin and held together by disulfide bridges. The autolysis of β-trypsin is cleaved at Lys131 and Ser132 in the bovine sequence.

Figure 1. The structure of trypsinogen (left) and trypsin (right). (Elgendy, A. S. 2016)

The structure of trypsin and chymotrypsin are very similar, but there is one important difference between the two structures. The specificity pocket of chymotrypsin contains a serine at the position 189 while trypsin includes an aspartic acid at that position. Based on the structural difference, chymotrypsin chooses the aromatic amino acids, which are hydrophobic, while trypsin chooses the basic amino acids, which contain a positive charge.

Mechanism

Trypsin can catalyze the hydrolysis reaction to break down peptides into amino acids, which shares a general catalytic mechanism with other serine proteases. The active site of trypsin is composed of three amino acids, which are serine 195, histidine 57, and aspartate 102, called a catalytic triad. The steps of catalytic process of trypsin include:

• Substrate binding. The amino acid side chain residue of substrate can bind to the active site on the trypsin, with the carbonyl carbon of this bond positioned near the nucleophilic serine 195.
• Nucleophilic attack. Histidine 57 facilitate the hydroxyl group of the nucleophile serine 195 abstracts a proton to attack the carbonyl carbon. A covalent bond is generated between the Ser195 side-chain and the substrate due to the nucleophilic attacks. A hydrogen bond stabilizes the negative charge that develops on the peptide carbonyl oxygen formed from two protease backbone amide protons.
• Protonation. The substrate amide nitrogen gets donated a hydrogen by His57 allowing release of the Carboxyl-terminal part of the substrate as a free peptide.
• Ester hydrolysis. The final step is the attack by water on the ester bond between the peptide and the Ser195 oxygen. Peptide with a normal carboxyl group a second product, and regenerates the serine hydroxyl. The second peptide then dissociates from the enzyme to allow another catalytic cycle begin.

Figure 2. Trypsin catalytic mechanism.

Physiological Functions

Trypsin is an important digestive enzyme present in pancreatic juices. The pancreas releases trypsinogen, chymotrypsinogen, and procarboxypeptidase when protein reaches the duodenum. Once trypsinogen touches enterokinase, which is secreted by the mucosal cells of the small intestines via the pancreatic duct then trypsin is activated. Then, trypsin is responsible for activating zymogens of other digestive enzymes, including procarboxypeptidase and chymotrypsinogen, which are converted into chymotrypsin and carboxypeptidase. In addition, trypsin can hydrolyze peptides into smaller forms of amino acids. Therefore, trypsin mainly functions as a hydrolyzing biological catalyst for peptide bonds.

Applications

Due to the trypsin is available easily in high quantity and high purity, it has been widely used in various biological technologies. During cell culture, trypsin is used to resuspend adherent cells during the process of cell passage and harvesting cells. Trypsin can also be used to dissociated dissected cells prior to cell fixing and sorting. Trypsin can break down the casein in breast milk, resulting in the milk to become translucent, which can be used to measure the rate of reaction. In biological research, trypsin is commonly used digest protein into peptides during proteomics experiments for mass spectrometry analysis. In the medical field, trypsin can also be used to dissolved blood clots and treat inflammation.

Reference

1. Elgendy, A. S., Abdelrasool, M. K. A literature review on Trypsin Enzyme. 2016.