Mechanism of Catalysis
The active site features a charge-relay network involving Asp-32, His-64, and active site Ser-221 arranged in a catalytic triad. The charge-relay network functions as follows: The carboxylate side-chain of Asp-32 hydrogen-bonds to a nitrogen-bonded proton on His-64's imidazole ring. This is possible because Asp is negatively charged at physiological pH. The other nitrogen on His-64 hydrogen-bonds to the O-H proton of Ser-221. This last interaction results in charge-separation of O-H, with the oxygen atom being more nucleophilic. This allows the oxygen atom of Ser-221 to attack incoming substrates (i.e., peptide bonds), assisted by a neighboring carboxyamide side-chain of Asn-155.
Even though Asp-32, His-64, and Ser-221 are sequentially far apart, they converge in the 3D structure to form the active site.
To summarize the interactions described above, Ser-221 acts as a nucleophile and cleaves peptide bonds with its partially negative oxygen atom. This is possible due to the nature of the charge-relay site of subtilisin.
Read more about this topic: Subtilisin
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