Design of activated serine–containing catalytic triads with atomic-level accuracy

摘要

Serine hydrolases use a conserved nucleophilic serine to hydrolyze ester, amide or thio-ester bonds in proteins and small molecules and constitute one of the largest enzyme families in the human proteome~1. The canonical serine-histidine–aspartic acid catalytic triad mechanism has been well studied: the serine Oγ is the nucleophile, the imidazole ring of the histidine acts as a general acid/base, and the carboxylate of the aspartic acid orients the imidazole ring and neutralizes the charge developed on the histidine in the transition state. In addition to the catalytic triad residues, a highly conserved oxyanion-binding site, commonly referred to as the oxyanion hole, stabilizes the negative charge on the carbony~l oxygen of the tetrahedral intermediate. The peptide backbone NH groups stabilize these high-energy species in most cases~(2–7). The activated nucleophilic serine can be covalently modified by electrophilic organophosphate compounds, resulting in the loss of catalytic activity. This has inspired the development of fluorophosphonates as activity-based probes for serine hydrolases, which enable monitoring of the activity of many serine hydrolases in parallel directly in complex proteomes by the activity-based protein profiling (ABPP) technology~(8,9).