Anthony Noles Sandbox 1
Mechanism of TrypsinMechanism of Trypsin
Trypsin, a protease, has great specificity and is therefore the most valuable member of the arsenal of proteases used to fragment polypeptide. It cleaves peptide bonds on the C side (toward the carboxyl terminus) of the positively charged residues Arg and Lys if the next residue is not Pro. More specifically, it is a member of the serine proteases, named for the common catalytic mechanism involving a peculiarly reactive Ser residue. As with other digestive enzymes chymotrypsin and elastase, trypsin catalyze the hydrolysis of peptide (amide) bonds but with different specificities for the side chains flanking the peptide bond. Trypsin is specific for a positively charged residue.
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| 2agi, resolution 1.14Å () | |||||||||
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| Ligands: | , | ||||||||
| Non-Standard Residues: | , | ||||||||
| Activity: | Trypsin, with EC number 3.4.21.4 | ||||||||
| Related: | 2age, 2agg, 2ah4 | ||||||||
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| Resources: | FirstGlance, OCA, RCSB, PDBsum | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
The primary structure of trypsin, PDB id 2agi, is approximately 240 residues. It has a reactive Ser and a catalytically essential His. The protein is folded into two domains, both of which have extensive regions of antiparallel beta-sheets in a barrel-like arrangement but contain little helix. There are catalytically essential residues, His 57 and Ser 195, which are located in the enzyme’s substrate-binding site. Asp 102 is buried in a nearby pocket, These three residues form a hydrogen bonded constellation referred to as the catalytic triad. The cationic side chains of trypsin’s preferred residues, Arg and Lys, can form ion pairs with this Asp residue.[1]
There is a relatively complex catalytic mechanism. Once trypsin has bound a substrate, Ser 195 nucleophilically attacks the peptides carboxyl group to form a tetrahedral intermediate. Ser 195 is ideally situated to nucleophilically attack due to proximity and orientation. This attack transfers a proton to the imidazole ring of His 57, forming imidazolium ion. This is aided by Asp 103, which is hydrogen bonded to His 57. The tetrahedral intermediate decomposes to the acyl-enzyme intermediate due to proton donation from N3 of His 57. The amide group is released from the enzyme and replaced by water. The acyl-enzyme intermediate is highly susceptible to hydrolytic cleavage, and adds water causeing a second tetrahedral intermediate. Reversing the initial nucleophilic attack on the substrate yields the carboxylate product, regenerating the active enzyme. For this step, water is the attacking nucleophile and Ser 195 is the leaving group.