How does trypsin digest protein?

Trypsin is a serine protease enzyme that specifically cleaves peptide bonds on the carboxyl side of lysine or arginine residues, except when either residue is followed by proline. This specific cleavage property of trypsin makes it a valuable tool in protein chemistry, particularly in protein sequencing and proteomics. Here's a step-by-step explanation of how trypsin digests proteins:

Binding: Trypsin binds to the surface of the protein substrate through specific interactions between its active site and the amino acid residues surrounding the cleavage site. This binding induces conformational changes that facilitate the catalytic reaction.

Catalysis: The active site of trypsin contains a catalytic triad consisting of three amino acid residues: histidine, aspartic acid, and serine. These residues work together to facilitate the hydrolysis (cleavage) of the peptide bond.

Histidine: Histidine acts as a proton donor, transferring a hydrogen ion (H+) to the carbonyl oxygen of the scissile peptide bond. This protonation weakens the bond, making it more susceptible to nucleophilic attack.

Aspartic acid: Aspartic acid functions as a general base, abstracting a proton from the hydroxyl group of the serine residue. This deprotonation generates a highly reactive serine hydroxyl group, which acts as the nucleophile in the reaction.

Serine: The activated serine hydroxyl group attacks the carbonyl carbon of the scissile peptide bond, forming a tetrahedral intermediate. This intermediate subsequently collapses, resulting in the cleavage of the peptide bond and the release of two peptide fragments.

Specificity: Trypsin exhibits specificity in its cleavage pattern due to the presence of a bulky side chain on its substrate-binding pocket. This bulky side chain prevents the binding of amino acids with large side chains (such as proline) next to the cleavage site. As a result, trypsin preferentially cleaves peptide bonds followed by lysine or arginine residues, except when they are followed by proline.

By selectively cleaving specific peptide bonds, trypsin generates a set of smaller peptide fragments that can be further analyzed or separated for various purposes, such as protein identification, sequencing, and characterization.