Trypsin is a serine protease that works enzymatically by using a mixture of base, acid, and covalent catalysis. The protein uses serine in its active site to interact covalently with the substrate. To create a nucleophilic attack, the histidine 57 group () activates the serine 195 group via base catalysis and covalent catalysis follows. To complete the formation of a nucleophile, aspartic acid 102 pulls positive charge from histidine 57, completing the and forming an effective nucleophile. This forms a tetrahedral intermediate, in which the anionic carbonyl oxygen moves into the active site to a location known as the oxyanion hole [1]. The tetrahedral intermediate is followed by acid catalysis from the -NH2 of the of the c-terminus, resulting in a broken peptide bond in the substrate.The transition state from the tetrahedral intermediate is stabilized by Asp 189 interacting with Gly 219 to create a stable . The acyl-enzyme intermediate is present, and hydrolysis occurs which ultimately releases the c-terminal chain of the substrate and forms a new bond between water and the carbonyl carbon of the enzyme-substrate complex. The covalent C-O bond of the substrate-enzyme complex is broken, and the enzyme is reformed as the product is released [2]
