Design and Production of an Organophosphorus-Bioscavenger Via Protein Engineering of the Human Acetylcholinesterase

摘要

The design of novel biocatalysts with potential pharmacological use against organophosphate (OP) poisoning, within the mold of recombinant human acetylcholinesterase (rHuAChE), was carried out by two complementing research approaches: (a) Kinetic studies combined with mass spectrometric analysis of rHuAChE and selected mutants thereof, with a variety of OPs for better understanding of the chemical environment for the OPs in the active center of AChE. Specifically, here we report the resolution of some of the less understood reaction pathways of phosphylation, and 'aging' of the resulting acetylcholinesterase (AChE) conjugates involving phosphoroamidates (P-N agents) such as tabun and butyl-tabun. (b) Biochemical and pharmacological analyses of post-translation modifications responsible for the limited residence time of rHuAChE. An extensive structural MALDI-TOF analysis of the N-glycans of the native serum-derived bovine AChE and its recombinant counterpart, identified specific termini that could account for the observed differential behavior of these enzymes. Accordingly, we generated through a combination of biochemical and genetic manipulations, a series of recombinant enzyme products differing in sialylation as well as oligomerization. A unique hierarchical order of post- translation modifications that determine the circulatory residence time was revealed. Based on these studies a recombinant product exhibiting a pharmacodynamic profile indistinguishable from that of the native enzyme was generated.